Pharmaceutical compositions

ABSTRACT

Novel methods for treating or reducing the likelihood of acquiring symptoms or diseases due to the menopause, in postmenopausal women, particularly osteoporosis, vaginal atrophy and dryness, hypogonadism, diminished libido, skin atrophy, connective tissue disease, urinary incontinence, breast, endometrial, ovarian and uterine cancers, hot flashes, loss of muscle mass, insulin resistance, fatigue, loss of energy, aging, physical symptoms of menopause, in susceptible warm-blooded animals including humans involving administration of a sex steroid precursor are disclosed. Said method comprising novel ways of administering and dosing dehydroepiandrosterone (DHEA) in order to take advantage of positive androgenic effects in the vaginal layers lamina propia and/or the layer muscularis, without undesirably causing systemic estrogenic effects in order to avoid the risk of breast and uterine cancer. Pharmaceutical compositions for delivery of active ingredient(s) useful to the invention are also disclosed.

FIELD OF THE INVENTION

The present invention provides novel ways of administering and dosingdehydroepiandrosterone (DHEA) in order to take advantage of positiveandrogenic effects (for example in the vaginal layers lamina propiaand/or the layer muscularis), without undesirably causing systemicestrogenic effects. In addition to DHEA, other sex steroid precursorsmay be used (e.g., dehydroepiandrosterone-sulfate,androst-5-ene-3β,17β-diol, and 4-androstene-3,17-dione).

BACKGROUND OF THE RELATED ART

Many hormone-related therapies are known. For example, many provide thesex steroids estrogen or androgen systemically and/or to target tissue.In addition to direct administration of androgens and/or estrogens, sexsteroid precursors that can be converted to estrogen and/or androgen ina given tissue have also been used for many conditions. Both androgensand estrogens can be beneficial in some contexts and detrimental inothers. That depends inter alia on the tissue being targeted, thespecific needs presented by a patient, and the extent to whichnon-targeted tissue may be affected. Some therapies, though targeted,can still have undesirable activity elsewhere in the body (e.g. wherelocal administration of the pharmaceutical agent nonetheless results inincreased systemic presence of either the pharmaceutical or one of itsmetabolites. Also, the mechanism of action has not always been fullyunderstood, especially the relative contributions of androgens andestrogens.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to utilize specificdosages, formulations and modes of administration to better achieve thebeneficial effects of sex steroids and to better avoid their undesirableside effects.

In one aspect, the invention provides a method of treating and/orreducing the likelihood of acquiring vaginal diseases or conditionsrelated to hormonal imbalance in postmenopausal women, said methodcomprising administering a sex steroid precursor selected from the groupconsisting of dehydroepiandrosterone, dehydroepiandrosterone-sulfate,androst-5-ene-3β,17β-diol, and 4-androsten-3,17-dione to a patient inneed of said treatment wherein the said sex steroid precursor isadministered at a therapeutic amount which increases the level ofcirculating androgen metabolites without increasing the level ofestradiol above the values found in normal postmenopausal women.

In another aspect, the invention provides a method of treating and/orreducing the likelihood of acquiring symptoms or diseases due to themenopause, in postmenopausal women said method comprising administeringa sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate,androst-5-ene-3β,17β-diol, and 4-androsten-3,17-dione to a patient inneed of said treatment wherein the said sex steroid precursor isadministered at a therapeutic amount which increases the level ofcirculating androgen metabolites without increasing the circulatinglevel of estradiol above the values found in normal postmenopausal womenin order to avoid the risk of breast and uterine cancer.

In another aspect, the invention provides a method of treating and/orreducing the likelihood of acquiring symptoms or diseases due to themenopause, in postmenopausal women, said method comprising administeringa sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate,androst-5-ene-3β,17β-diol, and 4-androsten-3,17-dione to a patient inneed of said treatment wherein the said sex steroid precursor isadministered at a therapeutic amount which increases the level ofcirculating androgen metabolites and further comprising administering aspart of a combination therapy, a therapeutically effective amount of aSelective Estrogen Receptor Modulator in order to avoid the risk ofbreast and uterine cancer normally present in postmenauposal women andto prevent bone loss, fat accumulation and diabetes type 2.

In another aspect, the invention provides method of treating vaginalconditions of the layer lamina propia or layer muscularis comprisingvaginal administration of DHEA in a daily dose of 343 mg.

In another aspect, the invention provides a pharmaceutical compositioncomprising a sex steroid precursor selected from the group consisting ofdehydroepiandrosterone, dehydroepiandrosterone-sulfate,androst-5-ene-3β,17β-diol, and 4-androstene-3,17-dione and furthercomprising a pharmaceutically acceptable excipient, diluent or carrierselected from the group consisting of triglycerides of saturated fattyacids C2-C18 with varied portions of the corresponding partialglycerides (hard fat, Witepsol), butter, mixed triglycerides of oleic,palmitic, and stearic acids (cocoa butter), partially hydrogenatedcottonseed oil (Cotomar), hydrogenated fatty alcohols and esters(Dehydag Base I, Base II or Base III, may also contains glycerides ofsaturated fatty acids C12-C16), triglycerides from palm, palm kernel,and coconut oils with self-emulsifying glyceryl monostearate andpolyoxyl stearate (Fattibase), Hexaride Base 95, higher meltingfractions of coconut and palm kernel oil (Hydrokote), Rearrangedhydrogenated vegetable oils (S-70-XX95 and S-070-XXA), eutetic mixtureof mono, di-, triglycerides derived from natural vegetable oils(Suppocire), Tegester Triglycerides, Tween 61, triglycerides derivedfrom coconut oil (Wecobee), theobroma oil, semi-synthetic glycerides(Japocire, Ovucire), mixture of tri- di- and monoglycerides of saturatedfatty acids (Massa Estarinum) and a combination of the foregoing (seeAllen et al. 2008). Any vehicle including liquid in which DHEA and otherprecursors are soluble covers by this invention.

In another aspect, the invention provides a vaginal suppositorycomprising 0.25-2.00 percent, more specially 0.5 percent DHEA, by weightrelative to the total weight of the suppository, of DHEA, and furthercomprising a lipophilic excipient. Particularly suitable excipient iswitepsol H-15.

By providing the desired androgenic effects without estrogenic systemiceffects, systemic side effects of estrogen such as the increased risk ofbreast and endometrial cancers found with current estrogen-based localand systemic estrogen replacement therapies (Labrie, Cusan et al.Menopause, in press) can be avoided.

In addition to other forms of administering precursors, the inventionprovides vaginal suppositories and vaginal creams formulated withpreferred excipients and preferred concentrations of precursor.

Vaginal administration is preferred because local action may provide thedesired androgenic effects on desired vaginal layers at much lowerdosages than when otherwise administered. Dosing by other means ofadministration may also be utilized by altering the foregoing dosagesand concentrations for known variation between the methods ofadministration. The attending clinician should alter dosagesappropriately in accordance with individual patient response.

In preferred embodiments, the sex steroid precursor is DHEA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows serum Levels of DHEA and 5-Diol on Day 1 or Day 7 in 40-75Year-Old Postmenopausal Women Following Daily Administration of VaginalSuppositories Containing 0%, 0.5%, 1.0% or 1.8% of DHEA. Data areexpressed as means±SEM (n=9 or 10).

FIG. 2 shows Serum Levels of Testo and DHT on Day 1 or Day 7 in 40-75Year-Old Postmenopausal Women Following Daily Administration of VaginalSuppositories Containing 0%, 0.5%, 1.0% or 1.8% of DHEA (n=8). Data areexpressed as means±SEM (n=8 to 9). Testo levels from one patient in theplacebo group were excluded because of unexplained high levels of Testonot reflected in any other steroid.

FIG. 3 shows Serum Levels of E₁ and E₂ on Day 1 or Day 7 in 40-75Year-Old Postmenopausal Women Following Daily Administration of VaginalSuppositories Containing 0%, 0.5%, 1.0% or 1.8% of DHEA. Data areexpressed as means±SEM (n=9 or 10).

FIG. 4 shows Serum Levels of E₁-S and DHEA-S on Day 1 or Day 7 in 40-75Year-Old Postmenopausal Women Following Daily Administration of VaginalSuppositories Containing 0%, 0.5%, 1.0% or 1.8% of DHEA. Data areexpressed as means±SEM (n=9 or 10).

FIG. 5 shows Serum Levels of 4-Dione and ADT-G on Day 1 or Day 7 in40-75 Year-Old Postmenopausal Women Following Daily Administration ofVaginal Suppositories Containing 0%, 0.5%, 1.0% or 1.8% of DHEA. Dataare expressed as means±SEM (n=9 or 10).

FIG. 6 shows Serum Levels of 3α-Diol-3G and 3α-Diol-17G on Day 1 or Day7 in 40-75 Year-Old Postmenopausal Women Following Once DailyAdministration of Vaginal Suppositories Containing 0%, 0.5%, 1.0% or1.8% of DHEA. Data are expressed as means±SEM (n=9 or 10).

FIG. 7 shows Average 24-Hour Serum Concentration (AUC_(0-24 h)/24) ofDHEA, 5-Diol, DHEA-S, 4-Dione, Testo and DHT Measured on Day 1 or Day 7Following Once Daily Administration of Vaginal Suppositories Containing0%, 0,5%, 1.0% or 1.8% of DHEA. Data are expressed as means±SEM (n=8 to10). Testo levels from one patient in the placebo group were excluded(n=8 in that group). Serum steroid concentrations measured in 30-35year-old premenopausal women are added as reference. Data are expressedas mean (n=47) while the 5^(th) and 95^(th) centiles are indicated(dashed lines). p<0.05, **, p<0.01, experimental (Day 7) versus placebo(Day 7).

FIG. 8 shows Average 24-Hour Serum Concentration (AUC_(0-24 h)/24) ofADT-G, 3α-Diol-3G, 3α-Diol-17G, E₁, E₂ and E₁-S Measured on Day 1 or Day7 Following Daily Administration of Vaginal Suppositories Containing 0%,0.5%, 1.0% or 1.8% of DHEA. Data are expressed as means±SEM (n=9 or 10).Serum steroid concentrations measured in 30-35 year-old premenopausalwomen are added as reference. Data are expressed as mean (n=47) whilethe 5^(th) and 95^(th) centiles are indicated (dashed lines). *, p<0.05,**, p<0.01, experimental (Day 7) versus placebo (Day 7).

FIG. 9 shows Changes of the Serum Levels of the Sum of the AndrogenMetabolites ADT-G, 3α-Diol-17G in Postmenopausal Women with VaginalAtrophy Following Intravaginal Administration of Increasing Doses ofDHEA. The data are expressed as percentage of the serum levels of thesame steroid metabolites observed in young adult (30-35 year-old)cycling premenopausal women. The level of transformation is obtained bydividing the sum of the serum levels of ADT-G, 3α-diol-3G and3α-diol-17G in women who received the 05%, 1.0% and 1.8% DHEA doses bythe values found in premenopausal women (data from Labrie et al., 2006).The serum DHEA changes compared to normal premenopausal women are alsoindicated as comparison to indicate efficiency of transformation(0 - - - 0). . . . ; and ______ basal levels of androgen metabolites andDHEA, respectively.

FIG. 10 shows Maturation Index (A) and Vaginal pH (B) Measured on Day 1and Day 7 in 40-75 Year-Old Postmenopausal Women Following DailyAdministration of Vaginal Suppositories Containing 0%, 0.5%, 1.0% or1.8% of DHEA. Data are expressed as means±SEM (n=9 or 10). *, p<0.05,**, p<0.01, Data on Day 7 versus Data on Day 1.

FIG. 11 shows a time-course of serum dehydroepiandrosterone (DHEA) (A)and androst-5-ene-3β,17β-diol (5-diol) (B) following single oraladministration of two 50-mg capsules of DHEA or the application of 4 gof 10% DHEA cream or gel to postmenopausal women.

FIG. 12 shows a time-course of serum androstenedione (4-dione) (A) andtestosterone (B) following single oral administration of two 50-mgcapsules of DHEA or the application of 4 g of 10% DHEA cream or gel topostmenopausal women.

FIG. 13 shows a time-course of serum estrone (E₁) (A) and 17β-estradiol(E₂) (B) following single oral administration of two 50-mg capsules ofDHEA or the application of 4 g of 10% DHEA cream or gel topostmenopausal women.

FIG. 14 shows a time-course of serum dehydroepiandrosterone sulfate(DHEA-S) (A) and estrone sulfate (E₁-S) (B) following single oraladministration of two 50-mg capsules of DHEA or the application of 4 gof 10% DHEA cream or gel to postmenopausal women.

FIG. 15 shows a time-course of serum androserone glucuronide (ADT-G) (A)and androstone 3α,17β-diol-glucuronide (3α-diol-G) (B) following dailyoral administration of two 50-mg capsules of DHEA or the application of4 g of 10% DHEA cream or gel to postmenopausal women.

FIG. 16 shows a time-course of serum dehydroepiandrosterone (DHEA) (A)and andros-5-ene-3β,17β-diol (5-diol) (B) following daily oraladministration of two 50-mg capsules of DHEA or the application of 4 gof 10% DHEA cream or gel to postmenopausal women. Measurements were madeon the 14th day of dosing.

FIG. 17 shows a time-course of serum androstenedione (4-dione) (A) andtestosterone (B) following daily oral administration of two 50-mgcapsules of DHEA or the application of 4 g of 10% DHEA cream or gel topostmenopausal women. Measurements were made on the 14th day of dosing.

FIG. 18 shows a time-course of serum estrone (E₁) (A) and estradiol (E₂)following daily oral administration of two 50-mg capsules of DHEA or theapplication of 4 g of 10% DHEA cream or gel to postmenopausal women.Measurements were made on the 14th day of dosing.

FIG. 19 shows a time-course of serum dehydroepiandrosterone sulfate(DHEA-S) (A) and estrone sulfate (E-S) (B) following daily oraladministration of two 50-mg capsules of DHEA or the application of 4 gof 10% DHEA cream or gel to postmenopausal women. Measurements were madeon the 14th day of dosing.

FIG. 20 shows a time-course of serum androsterone glucuronide (ADT-G)(A) and androstene-3β,17β-diol-G (3α-diol-G) (B) following daily oraladministration of two 50-mg capsules of DIEA or the application of 4 gof 10% DHEA cream or gel to postmenopausal women. Measurements were madeon the 14th day of dosing.

FIG. 21 shows ratios of the AUC_(0-24 h) values of DHEA and itsmetabolites on the 14th day of dosing compared to the pretreatment basalvalues. The corresponding numerical values can be found in Table 5.

FIG. 22 shows the effect of daily intravaginal application of 0.0%,0,25%, 0.5% and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on thepercentage of vaginal parabasal cells in postmenopausal women. Data areexpressed as means 3 SEM.

FIG. 23 shows the effect of daily intravaginal application of 0.0%,0.25%, 0.5% and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on thepercentage of vaginal superficial cells in postmenopausal women. Dataare expressed as means 3 SEM.

FIG. 24 shows the effect of daily intravaginal application of 0.0%,0.25%, 0.5% and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks onvaginal pH in postmenopausal women. Data are expressed as means±SEM.

FIG. 25 shows the effect of daily intravaginal application of 0.0%,0.25%, 05% and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on thechange in severity of the symptom of vaginal atrophy judged by womenthemselves as being the most bothersome. Values are compared to day 1and are expressed as means SEM.

FIG. 26 shows the effect of daily intravaginal application of 0.0%,0.25%, 0.5% and 1.0% DIEA (Prasterone) for 2, 4, 8 and 12 weeks on thechange in vaginal secretions evaluated at vaginal examination. Data areexpressed as means±SEM.

FIG. 27 shows the effect of daily intravaginal application of 0.0%,0.25%, 0.5% and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on thechange in vaginal color evaluated at vaginal examination, Data areexpressed as means±SEM.

FIG. 28 shows the effect of daily intravaginal application of 0.0%,0.25%, 0.5% and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on thechange in vaginal epithelial integrity evaluated at vaginal examination.Data are expressed as means±SEM.

FIG. 29 shows effect of daily intravaginal application of 0.0%, 0,25%,0.5% and 1.0% DHEA (Prasterone) for 2, 4, 8 and 12 weeks on the changein vaginal epithelial thickness evaluated at vaginal examination. Dataare expressed as means±SEM.

FIG. 30 shows the average 24-hour serum concentrations (AUC_(0-24 h)/24)of DHEA, 5-Diol, DHEA-S, E₁, E₂ and EA-S measured on days 1 and 7following once daily administration of vaginal ovule containing 0.5%DHEA. Data are expressed as means t SEM (n=10). Serum steroidconcentrations measured in 30-35 year-old premenopausal (n=47) as wellas in 55-65 year-old postmenopausal (n=369) women are added as referencedata which are expressed as means and 5^(th) and 95^(th) centiles(dashed lines). p<0.05,**, p<0.01, experimental versus baseline. (Dataare from Labrie, Cusan et al. 2008),

FIG. 31 shows the average 24-hour serum concentrations (AUC_(0-24 h)/24)of 4-Dione, testosterone, DHT ADT-G, 3α-Diol-3G and 3α-Diol-17G measuredon days 1 and 7 following once daily administration of vaginal ovulecontaining 0.5% DHEA. Data are expressed as means±SEM (n=10). Serumsteroid concentrations measured in 30-35 year-old premenopausal (n=47)and 55-65 year-old postmenopausal (n=369) women are added as referencedata which are expressed as means and 5^(th) and 95^(th) centiles(dashed lines). *, p<0.05, experimental versus baseline. (Data are fromLabrie, Cusan et al. 2008).

DETAILED DESCRIPTION OF THE INVENTION

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Vaginal dryness is found in 75% of postmenopausal women (Wines andWillsteed 2001; N.A.M.S. 2007). For various reasons, especially the fearof complications by estrogens, only 20 to 25% of symptomatic women withvaginal atrophy seek medical treatment (Pandit and Ouslander 1997;N.A.M.S. 2007). There is thus a clear medical need and a majoropportunity to improve the quality of life of a large population ofwomen left suffering from vaginal atrophy for a large proportion oftheir lifetime. In can be mentioned that while hot flashes abatespontaneously with time, vaginal atrophy symptoms, namely vaginaldryness, vulvovaginal irritation/itching and dyspareunia usuallyincrease in severity with time in the absence of treatment.

Based upon the well known fact that estrogen secretion by the ovariesceases at menopause, systemic and local estrogens have so-far been theexclusive approach for the treatment of vaginal atrophy, However,systemic estrogens+progestin (14R) and estrogens alone (ERT) have beenshown to increase the risk of breast cancer (Steinberg, Thacker et at1991; Sillero-Arenas, Delgado-Rodriguez et al. 1992 Colditz, Egn et al1993; Colditz, Hankinson et at 1995; Collaborative Group on HormonalFactors in Breast Cancer 1997; Hulley 2002; Beral 2003; Chlebowski,Hendrix et at 2003; Holmberg and Anderson 2004; Lytinen, Pukkala et at2006; Corrao, Zambon et al, 2008; Holmberg, Iversen et al. 2008; Li,Plummer et al. 2008), ovarian cancer (Garg, Kerlikowske et al. 1998;Coughlin, Giustozzi et al. 2000; Lacey, Mink et al. 2002; Riman, Dickmanet al. 2002; Rodriguez, Patel et al, 2002; Rossouw, Anderson et al. 20Z;Lyytinen, Pukkala et al. 2006) as well as endometrial cancer (estrogensalone) (Gambrell, Massey et al. 1980; Persson, Adami et al. 1989; Voigt,Weiss et al. 1991; Jick, Walker et al. 1993; Grady, Gebretsadik et al,1995; Beral, Bull et al. 2005). The publicity which followed the Women'sHealth Initiative Study (Rossouw, Anderson et al. 2002) had the greatestimpact, thus putting in doubt the safety of the available treatments ofmenopausal symptoms (Archer 2007).

Although intravaginal formulations were developed to avoid systemicexposure to estrogens, a long series of studies have unanimouslydemonstrated that all intravaginal estrogen formulations lead torelatively high serum estrogen levels measured directly or through theirsystemic effects (Englund and Johansson 1978; Rigg, Hermann et al. 1978;Martin, Yen et al, 1979; Furuhjelm, Karlgren et al, 1980; Deutsch,Ossowski et al. 1981; Mandel, Geola et al. 1983; Nilsson and Heimer1992: Nachtigall 1995; Ayton, Darling et al. 1996; Dugal, Hesla et al.2000; Rioux, Devin et al. 2000; Manonai, Theppisai et al. 2001;Notelovitz, Funk et al. 2002; Ponzone, Biglia et al. 2005; Weisberg,Ayton et al. 2005; Galhardo, Soares et al. 2006; Kendall. Dowsett et al.2006; Long, Liu et al. 2006; Bachmann, Lobo et al. 2008). These datashowing a significant increase in serum estrogen levels clearly indicatethat the use of intravaginal estrogen formulations is also potentiallyassociated with an increased risk of breast and uterine cancer (Kvorningand Jensen 1986; Mattson, Culberg et al. 1989; Rosenberg, Magnusson etal. 2006; N.A.M.S. 2007). Concerns have in fact been officially raisedabout the stimulatory effects of vaginal estrogen formulations on theendometrium ((N.A.M.S. 2007).

Most previous measurements of the serum estradiol (E₂) levels afterintravaginal administration of estrogens used radioimmunoassays, atechnology lacking specificity, accuracy, reliability and sensitivity(Rinaldi, Dechaud et al. 2001). We have measured serum estrogens usingGLP (Good Laboratory Practice)-validated mass spectrometry assaysfollowing intravaginal administration of the two most commonly usedestrogen formulations (Labrie, Cusan et al. 2008). This study coulddefinitively show that both the E₂ pill (2=g E₂/day) and conjugatedestrogens cream (1 g of 0.625 mg conjugated estrogens/day), afterone-week of daily treatment, cause an approximately 5-fold increase inserum E₂ in postmenopausal women. Such data indicate that the effects ofestrogens applied locally in the vagina are unlikely to be limited tothe vagina and that systemic action is expected as previously suggested(Englund and Johansson 1978; Rigg, Hermann et al. 1978; Martin, Yen etal. 1979; Furuhjelm, Karlgren et al. 1980; Deutsch, Ossowski et al.1981; Mandel, Geola et al. 1983; Nilsson and Heimer 1992; Nachtigall1995; Ayton, Darling et al. 19%; Dugal, Hesla et at 2000; Rioux, Devlinet al. 200; Manonai, Theppisai et al. 2001; Notelovitz, Funk et al.2002; Ponzone, Biglia et al. 2005; Weisberg, Ayton et al. 2005;Galhardo, Soares et a 2006; Kendall, Dowsett et al. 2006; Long, Liu etal, 2006; Bachmann, Lobo et al. 2008).

In addition to the above-indicated safety concerns of estrogensadministered both systemically and locally, recent data have clearlydemonstrated that women are not only deficient in estrogens at time ofmenopause but that they have also been progressively deprived, startingin the thirties, from the androgens made in specific peripheral targettissues by the intracrine transformation of dehydroepiandrosterone(DHEA) into androgens and/or estrogens (Labrie, Bélanger et al. 1988;Labrie 1991; Labrie, Luu-The et al. 2003; Labrie, Luu-The et al. 2005).In fact, serum DHEA and DHEA-sulfate progressively decrease from thepeak seen at the age of 30 years (Orentreich, Brind et al. 1984; Labrie,Bélanger et al. 1997; Labrie, Luu-The et al. 2003) to a value 60% lowerat time of menopause (Labrie, Bélanger et al. 2006).

Concerning the role of androgens in women, it is important to mentionthat women secrete 50% as much androgens as observed in men (Labrie,Bélanger et al. 1997; Labrie, Luu-The et al. 2005). Since serum DHEA isthe predominant source of androgens which play a series of physiologicalroles in women (Labrie, Luu-The et al. 2003; Labrie 2007). the 60%decrease in circulating DHEA already found at time of menopause leads toa similar 60% decrease in the total androgen pool (Labrie, Bélanger etal. 2006) with the resulting potential signs and symptoms ofhypoandrogenicity in the bone, muscle, skin, mammary gland, vagina,brain as well as on glucose, insulin and lipid metabolism (Labrie,Luu-The et al. 2003; Labrie 20). Among the androgen target tissues,recent data have shown that the vagina is sensitive to androgensfollowing DHEA administration in the rat with beneficial effects, notonly on the superficial epithelial layer of the vagina but also oncollagen fibers in the lamina propria and on the muscularis (Berger,El-Alfy et al. 2005).

).Based upon the data of our preclinical (Sourla, Flamand et al. 1998;Berger, El-Alfy et al, 2005) and clinical (Labrie, Diamond et al, 1997;Labrie, Cusan et al. 2008) studies showing beneficial effects on thevagina of DHEA administered percutaneously or locally, the presentclinical trial is a prospective, randomized and placebo-controlled studyof the effect of three doses of intravaginal DHEA administered daily for12 weeks on the changes in superficial and parabasal cells, vaginal pHand the most bothersome symptom of vaginal atrophy as primaryobjectives. The data clearly show that locally administered DHEA is veryefficient and rapid in correcting all the signs and symptoms of vaginalatrophy, a near maximal effect being already achieved at 2 weeks at aDHEA dose causing no significant change in serum estrogens or androgenswhile all other steroids remain unchanged or well within the range foundin normal postmenopausal women.

When DHEA is administered locally in the vagina, the beneficial effectsof estrogens and androgens made locally in the vagina are achievedwithout any significant release of estradiol or testosterone into theblood (Labrie, Cusan et al. J. Ster. Biochem. Mol. Biol. In press). Inthe formation of androgens and/or estrogens from DHEA by the process ofintracrinology, any tissue is unpredictable because the response dependsupon the activity of the enzymatic machinery specifically present ineach cell of each tissue. Thus, it is not possible to predict, from theandrogens and estrogens that are produced from DHEA in one tissue, theextent to which similar androgens and estrogens may be produced inanother tissue.

The results of the clinical trial ERC-210 (Example 3) clearlydemonstrate for the first time that the local administration of DHEA ashormone precursor replacement therapy (HPRT) is highly efficient andrapid in correcting all the symptoms and signs of vaginal atrophy inpostmenopausal women. Most importantly, this is achieved at a dose(0.5%) of DHEA which does not increase the serum levels of activeestrogens or androgens and with no or minimal changes in serum DH EA andany of its metabolites which all remain well within the range of valuesfound in normal postmenopausal women (Labrie, Cusan et al. 2008).

While 75% of postmenopausal women suffer from vaginal atrophy (Wines andWillsteed 2001; N.A.M.S. 2007), thus affecting their quality of lifeduring a major part of their lifetime, only 20% seek treatment (Panditand Ouslander 1997). The fear of breast cancer related to increasedblood levels of estrogens is the main reason involved. Since estrogensecretion into the systemic circulation is exclusively of ovarian originand ceases at menopause, administration of estrogens to postmenopausalwomen does not appear to be physiological. In the aftermath of WHI, thescientific challenge is to explore alternative hormonal therapy typesand formulations that would provide all the menopausal advantages ofestrogens while improving women's quality of life, minimizing risks andmaximizing benefits (Archer 2007). Since the non-estrogen basedtreatments have not shown convincing efficacy (Nelson, Vesco et a. 2006;Suckling, Lethaby et al. 2006), women and their physicians are left withno safe treatment for vaginal atrophy.

Various forms of estrogens are an efficient treatment for vulvovaginalatrophy (Pandit and Ouslander 1997; Utian, Shoupe et al. 201). In fact,the vaginal E₂ tablet has shown an efficacy similar to the E₂ ring(Weisberg, Ayton et al. 2005) as well as to the conjugated estrogencream (Rioux, Devlin et a). 2000; Manonai, Theppisai et al. 2001).

This novel HPRT is in marked contrast with the 5-fold increase in serumE₂ measured by mass spectrometry after treatment with intravaginal E₂ orconjugated estrogens (Labrie, Cusan et al 2008). These recent data onthe changes in serum estrogens confirm a long series of studies showingthat all intravaginal estrogen formulations lead to elevated serumestrogen concentrations measured directly by radioimmunoassays orthrough their systemic effects (Englund and Johansson 1978; Rigg,Hermann et al, 1978; Martin, Yen et al. 1979; Furuhjelm, Karlgren et al,1980; Deutsch, Ossowski et al. 1981; Mandel, Geola et al 1983; Nilssonand Heimer 1992; Nachtigall 1995; Ayton, Darling et al. 1996; Dugal,Hesla et al, 2000; Rioux, Devlin et al. 2000; Manonai, Theppisai et at2001; Notelovitz, Funk et al. 2002; Ponzone, Biglia et at 2005;Weisberg, Ayton et al, 2005; Galhardo, Soares et al. 2006; Kendall,Dowsett et al. 2006; Long Liu et al. 2006; Bachmann, Lobo et al, 2008).

The most common adverse events reported with vaginal estrogens arevaginal bleeding and breast pain, both secondary to increased serumestrogens (Suckling, Lethaby et al. 2006). These side effects have beenreported for the E₂ ring, conjugated estrogens cream as well as E₂tablet (Ayton, Darling et al. 1996; Weisberg, Ayton et al. 2005). Asmentioned above, concerns also exist about the stimulatory effects ofvaginal estrogens on the endometrium (N.A.M.S. 2007). Uterine bleeding,breast pain and perineal pain were reported in 9% of women who took thevaginal tablet for 24 weeks while 34% complained of the same symptoms inthe vaginal conjugated estrogen cream group (Rioux, Devlin et al. 2000).(Suckling, Lethaby et al. 2006) reported no difference between thedifferent vaginal estrogen preparations.

t is well known that atrophic vaginitis in postmenopausal women can beworsened or induced by the use of aromatase inhibitors for the treatmentof breast cancer. In fact, these drugs exert their benefits on breastcancer by decreasing E₂ biosynthesis in all tissues, thus increasing thefrequency and severity of menopausal symptoms (Fallowfield, Cella et al.2004; Morales, Neven et al. 2004). In a recent study where seven breastcancer patients treated with aromatase inhibitors received Vagifem at adaily dose of 25 μg for 2 weeks and then, thereafter, twice weekly,serum E₂ rose from a median of 3 pmol/l to 72 pmol/l, at 2 weeks (range3 to 232) (Kendall, Dowsett et al, 2006). Serum E₂ levels generallydecreased thereafter to values of 40 pmol/l or less although values of137 and 219 pmol/l were found at weeks 7-10. A patient who receivedPremarin cream had serum E₂ levels of 83 pmol/l at 2 weeks. It should bementioned that blood sampling for E₂ measurement was performed at timeof patient's visit, a timing not likely to correspond to the highestserum E levels after Vagifem administration. It is thus more than likelythat the values reported in (Kendall, Dowsett et al. 2006)underestimate, up to an unknown extent, the true elevation of serum E₂after intravaginal Vagifem pill or Premarin cream administration. Theauthors concluded that the use of Vagifem with aromatase inhibitors iscontraindicated. These findings obtained in breast cancer women treatedwith aromatase inhibitors raise a serious issue about the use of anyvaginal as well as any oral or transdermal estrogen preparation inpostmenopausal women.

The relatively high elevation of serum E₂ following treatment withvarious vaginal estrogen preparations leading to increased risk ofbreast cancer is a well recognized issue (Rosenberg, Magnusson et al.2006). Although a study having a small number of events and a shortfollow-up (a 4.7% subgroup among 1472 women) did not find astatistically significant difference in disease-free survival in thesubgroup of women who used vaginal estrogen (Dew, Wren et al. 2003), itdoes not appear reasonable or acceptable to increase the serum E₂ levelsduring breast cancer therapy when the objective of treatment witharomatase inhibitors is precisely to achieve the maximal inhibition ofE₂ biosynthesis.

In an early study with Vagifem, a E₂ tablet, when administered at the 25μg dose, led to serum E₂ levels of 80 pmol/l with values below 50 pmol/lat 14 h and later (Kvorning and Jensen 1986). In a more recent studywith Vagifem, maximal and mean 24 h serum E₂ concentrations weremeasured at 180±99 pmol/l and 84 pmol/l for the 25 μg dose while valuesof 81±62 pmol/l and 40 pmol/l, respectively, were found for the 10 μgdose (Notelovitz, Funk et al. 2002). Other vaginal estrogen tablets andcreams have led to even higher serum estrogen levels (Schiff, Tulchinskyet al. 1977; Rioux, Devlin et al. 2000).

With the 10 μg and 25 μg E₂ vaginal tablets, serum E₂ was found toincrease to maximal values of approximately 90 and 160 pmol/l,respectively, from basal values of approximately 35 pmol/l (Nilsson andHeimer 1992). Serum E₂ with Vagifem has been reported at a Cmax of 51±34μg/m on day 1, this value being practically unchanged on days 14 (47±21pg/ml) and 84 (49±27 pg/ml) (Vagifem, Physician Package Insert 1999).

In another study, after 52 weeks of treatment with 25 μg Vagifem, theserum levels of E₂ were reported to have remained unchanged from10.3±21.5 pg/ml to 9.9 pg/ml (Bachmann, Lobo et al. 2008). Such data canbe explained by the fact that blood sampling was most likely performed 3or 4 days after Vagifem application. It is also important to mentionthat the elevated pretreatment serum E₂ levels in that study most likelyrelate to the lack of specificity of the immuno-based assays used sincenormal E₂ serum levels measured by mass spectrometry in postmenopausalwomen are two to three times lower (Labrie, Bélanger et al. 2006).

In an early study, the oral and vaginal administration of 1.25 mgPremarin led to serum levels of E₂ and estrone up to at least 100 pg/mland 1000 pg/ml respectively, during the 24 h following administration,the levels being somewhat higher after vaginal application. Serumgonadotropin levels were decreased in most subjects (Englund andJohansson 1978). Similar data were reported by (Rigg, Hermann et al.1978). In a recent study, following 3 months of daily oral orintravaginal administration of 0.625 mg Premarin, the serum E₂ levelsincreased to 83.1 and 58.6 pg/ml respectively (Long Liu et al 2006),thus illustrating the very important systemic exposure after bothintravaginal and oral estrogen administration since serum E₂ was only36% lower after intravaginal compared to oral administration ofconjugated estrogens. In a 12-week study with Premarin vaginal cream atthe daily 2 g dose, three times a week, 21% of women experiencedbleeding after a progestogen test (Nachtigall 1995). Moreover, of thesewomen, 12% showed an increase in endometrial thickness at echography.

No increase in serum E₁, E₂ or E₁S levels have been reported with theuse of the vaginal ring (Nachtigall 1995; Gupta. Ozel et al. 2008)although significant increases in E₁S and E₂ have been observed in womenolder than 60 years (Naesen, Rodriguez-Macias et al. 2001). In theESTring group of a recent study, serum E₂ increased from 16±22 pmol/l to49±64 pmol/l at week 24 (Weisberg, Ayton et al. 2005). In the Vagifemgroup, on the other hand, serum E₂ increased from 15±33 pmol/l to 36±51pmol/l. These authors, nevertheless, reported that serum E₂ remainedwithin or near the values found in normal postmenopausal women. At 48weeks of treatment with ESTring or Vagifem, 30-32% of women hadcomplaints of urinary frequency, 36-39% of urinary urgency and 18-33%complained of dyspareunia (Weisberg, Ayton et al, 2005).

Three studies have documented that the E₂ vaginal ring permits low serumE₂ during the 90-day period except for the burst in serum estrogen thatreaches the lower region of those seen in normal cycling women or 100 to200 pmoles/L during the first 0.5-8 h after insertion of the ring(Holmgren, Lindskog et al. 1989; Schmidt, Andersson et al, 1994) (Bakerand Jaffe 19%). That the daily delivery of 7.5 μg of E₂ by theintravaginal route has systemic effects is shown by the observation of asignificant increase in bone mineral density of total hip and lumbarspine after 2 years of treatment with such an intravaginal dose of E₂(Salminen, Saaf et al. 2007).

As mentioned above, concerns exist about the stimulatory effects ofvaginal estrogens on the endometrium (N.A.M.S. 2007). After 12 weeks oftreatment of 32 women with 25 μg of intravaginal Ea (Vagifem), onepatient had simple hyperplasia without atypia (Bachmann, Lobo et al,2008). In a 24-week study involving 80 women, one case of proliferativeendometrium was found (Rioux, Devlin et al. 2000) and in another 52-weekstudy of 31 women, two had a proliferative endometrium (Mettler andOlsen 1991).

In a 12-week study with Premarin vaginal cream at the dose of 2 g, threetimes a week, 21% of women experienced bleeding after a progestogen test(Nachtigall 1995). Of these, 12% showed an increase in endometrialthickness by echography. The use of a 0.3 mg dose of conjugatedestrogens administered intravaginally, three times a week, may induceendometrial proliferation, albeit rarely, since endometrialproliferation was seen in only one of twenty cases (Nachtigall 1995).

The sustained-releaset estradiol ring (ESTring) induced endometrialproliferation similar to the 0.625 mg Premarin cream (Ayton, Darling etal. 1996) but less then the 1.25 mg Premarin cream (Nachtigall 1995). Infact, both the vaginal ring (ESTring) and conjugated estrogen cream(Premarin cream) have been shown to induce endometrial proliferation(Nachtigall 1995; Ayton, Darling et al, 1996). Two cases of moderateendometrial proliferation or hyperplasia in an endometrial polyp werefound with the E₂ ring (Nachtigall 1995), while two cases of hyperplasia(one simple and one complex, without atypia) were found with theconjugated estrogen cream in a trial of conjugated estrogen cream versusE₂ tablet (Rioux, Devlin et al. 2000), The E₂ vaginal tablet has beenassociated with endometrial hyperplasia similar to the estriol vaginaltablet (Dugal, Hesla et al. 2000; Manonai, Theppisai et al. 2001) butless than the conjugated estrogens cream (Manonai, Theppisai et al.2001).

Although serum estrogen levels are increased to a lower degree followinglocal intravaginal application compared to oral or percutaneous HRT orERT, the risk of breast cancer remains an issue and the safety of theintravaginal estrogens is in doubt (Suckling, Lethaby et al. 2006;N.A.M.S. 2007). In fact, although the increase in serum estrogens islower after the intravaginal compared to the oral or percutaneous routeof administration, it is significantly elevated above normalpostmenopausal levels for all intravaginal estrogen formulations(Ponzone, Biglia et al. 2005).

In addition to the increased breast cancer risk associated with theadministration of estrogens, it is important to remember that the truehormonal difference between the postmenopausal women who do not sufferfrom vaginal atrophy (estimated at 25% of the postmenopausal population)and the remaining 75% of postmenopausal women who suffer from vaginalatrophy (Wines and Willsteed 2001; N.A.M.S. 2007), is not related to thesecretion of estrogens in the systemic circulation since ovarianestrogen secretion has ceased in all women at time of menopause.Consequently, a deficit in estrogen secretion is not a valid explanationfor the occurrence of symptoms of vaginal atrophy in the majority ofpostmenopausal women.

Sex steroid formation, however, does not stop with the cessation ofovarian function at menopause. The recent progress in our understandingof the endocrine physiology in women show that after menopause, DHEAsecreted by the adrenals is the only source of sex steroids madeexclusively in target tissues (Labrie 1.991), Contrary to the estrogensof ovarian origin which are secreted in the general circulation wherethey can be measured, DHEA is an inactive precursor which is transformedin the peripheral tissues at various rates according the level ofexpression of the steroidogenic enzymes in each tissue. The process ofintracrinology permits local intratissular formation of active sexsteroids with no significant release of the active steroids in thecirculation (Labrie, Dupont et al. 1985; Labrie, Bélanger et al. 1988;Labrie 1991; Labrie, Luu-The et al. 2005).

The secretion of DHEA. however, decreases with age, a 60% decrease beingalready observed at time of menopause (Labrie, Luu-The et al. 2003;Labrie, Bélanger et a 2005; Labrie, Luu-The et al. 2005; Labrie, Bangeret al. 2006; Labrie, Luu-The et al. 2006; Labrie 2007). The onlydifference between the symptomatic and the asymptomatic postmenopausalwomen is the amount of DHEA secreted by the adrenals or the sensitivityof the vaginal tissue to DHEA. The difference of sensitivity ofdifferent women is likely to be related up, to an unknown extent, to thelevel of activity of the enzymatic machinery specific to each cell typein each tissue (Labrie 1991; Labrie, Bélanger et al, 2005). With thisknowledge, DHEA and not estrogens is a physiological hormonalreplacement therapy for postmenopausal women.

As well demonstrated in our previous studies (Labrie 1991; Labrie,Luu-The et al. 2003; Labrie, Luu-The et al. 2005; Labrie, Bélanger etal., 2007), supplementation with physiological amounts of exogeneousDHEA permits the biosynthesis of androgens and/or estrogens only in theappropriate target tissues which contain the required steroidogenicenzymes of intracrinology (Labrie, Luu-The et al. 2005). The activeandrogens and estrogens synthesized locally from DHEA in peripheraltarget tissues exert their action in the same cells where theirformation takes place. Most importantly, very little leakage of theactive sex steroids into the circulation takes place, thus explainingthe marked beneficial effects observed in the vagina with no significantchange in circulating estrogens or androgens (Labrie, Cusan et al.2008). This local biosynthesis, action and inactivation of estrogens andandrogens in target tissues eliminates the exposure of other tissues toexcess sex steroids and thus eliminates the increased risks ofundesirable side effects from elevated estrogen exposure, includingbreast, ovarian and uterine cancer (Gambrell, Massey et al. 1980;Persson, Adami et al. 1989; Steinberg, Thacker et al. 1991; Voigt, Weisset al. 1991; Sillero-Arenas, Delgado-Rodriguez et al. 1.992; Colditz,Egn et al, 1993; Jick, Walker et al, 1993; Colditz, Hankinson et at1995; Grady, Gebretsadik et al. 1995; Collaborative Group on HormonalFactors in Breast Cancer 1997; Garg, Kerlikowske et al. 1998; Coughlin,Giustozzi et at 2000; Hulley 2002; Lacey, Mink et al. 2002; Riman,Dickman et al. 2002; Rodriguez, Patel et al. 2002; Rossouw, Anderson etal. 2002; Beral 2003; Chlebowski, Hendrix et at 2003; Holmberg andAnderson 2004; Beral, Bull et al. 2005; Lyytinen, Pukkala et al. 2006;Corrao, Zambon et at 2008; Holmberg, Iversen et al 2008; i., Plummer etat 2008).

Change in pH is now recognized as a valid parameter which reflects thebeneficial effect of vaginal atrophy therapy. After 12 weeks ofintravaginal treatment with 25 g E₂, the percentage of patients having apH less than 5. was 51% compared to 21% in the placebo group (Bachmann,Lobo et al. 2008). At baseline, however, 11.2% and 13% of women had a pHbelow 5.0 in the two corresponding groups. In the clinical trial ERC-210(Example 3), no patient had a pH below 5.0 at start of therapy and 12%,36%, 46% and 48% had pH values below 5.0 at 12 weeks in the 0%, 0.25%,0.5% and 1.0% DHEA groups, respectively.

In clinical trial ERC-210 (Example 3), the effect of DHEA on thematuration of the vaginal epithelial cells is particularly rapid: withthe 0.5% DHEA ovule, 79% of the maximal effect on parabasal cells wasalready observed at 2 weeks while 48% of the maximal stimulatory effectexerted on superficial cells was observed at the same time interval. Onthe other hand, 85% of the maximal effect of 05% DHEA on the percentageof superficial cells was achieved at 4 weeks. Similarly, 63% of themaximal effect of 0.5% DHEA on the most bothersome symptom was observedat 2 weeks and 87% was reached at 4 weeks. Moreover, only 178% of womenreported no change in their most bothersome symptom at 12 weeks in the0.5% DHEA group compared to 48.8% in the placebo group.

The effect of DHEA on parabasal cells is rapid since the % of parabasalcells was decreased to less than 20% at one month with the three DHEAdoses used. The effect on the % of superficial cells is also very rapidwith 100% of the effect being seen at 2 weeks with the high (1%) DHEAdose. In a study with vaginal estrogen cream or tablet, approximately50% of the effect measured at 12 weeks was observed at 2 weeks (Rioux,Devlin et al. 2000). Such data indicate that the rapidity of the effectof DHEA is not inferior and possibly superior to the effect of thevaginal E₂ and conjugated estrogen formulations.

In a study of the effect of oral estrogens in 71 postmenopausal women,daily administration of 0.3 mg oral synthetic conjugated estrogensdecreased parabasal cells from 23% to 2.3% while superficial cellsincreased from 21% to 15.9% (Marx, Schade et al, 2004. In a studycomparing the 0.3 mg and 0.625 mg does of conjugated equine estrogens(Utian, Shoupe et al. 2001), the 0.625 mg dose has shown a greatereffect on the % of superficial cells.

In a recent study, the vaginal maturation value (VMV) increased from27.45 at baseline to 56.85 (p<0.0001) in the estrogen-treated group(Simon, Reape et al. 2007). The percentage of superficial cellsincreased by 17.15 from baseline while the percentage of parabasal cellsdecreased by 41.66% in the estrogen-treated group. In the same study,the vaginal pH decreased from 6.74 at baseline to 5.05 (decrease of 1.69or 24%) in the estrogen group). The severity of the most bothersomesymptoms decreased from 2.58 to 1.04 (−1,54) in the estrogen groupcompared to a decrease from 2.59 to 1.84 (−0.75) in the placebo group.Such data observed with estrogens are comparable to the 1.56 decrease inseverity of the most bothersome symptoms at 12 weeks in the 0.5% DHEAgroup and the 0.67 decrease in the placebo group observed in clinicaltrial ERC-210 (Example 3).

At week 12, 11% of ESTring subjects and 24% of Vagifem subjects hadpersistent atrophic epithelium. At week 48, the respective values were8% and 14% (Weisberg, Ayton et al. 2005). At 48 weeks of treatment withVagifem or ESTring, vaginal dryness was still present in 33% of women(Weisberg, Ayton et al. 2005). Pruritus vulvae, on the other hand,remained present in 15% and 20% of women after treatment with EST ringand Vagifem, respectively while 33% and 28% of women still haddyspareunia after treatment with ESTring and Vagifem, respectively.Bleeding after the progestogen test was 7% in the Vagifem group and 0%in the ESTring group.

After 3 months of daily administration of 0.625 mg Premarin orally orintravaginally (cream), respective 70.6% and 75% improvements ofdyspareunia were observed (Long, Liu et al. 2006). It was concluded inthat study that 1 g of 0.625 mg Premarin was the minimal dose for thetreatment of sexual dysfunction.

In women who received 25 μg E₂ intravaginally, dyspareunia persisted in12.4% of cases after 12 months of treatment (Simunic, Banovic et al,2003). The success rate of therapy of local E₂ tablets was 84.5% asjudged by patients and 86.1% as judged by doctors (Simunic, Banovic etal. 2003). Bachman et al, 1992(Bachmann, Notelovitz et at 1992) havereported that 40-50% of women on oral estrogen replacement therapy hadpersistent complaints of vaginal dryness.

As reported previously after 12 months of treatment with DHEA (Labrie,Diamond et al, 1997), the clinical trial ERC-210 (Example 3) shows noeffect on endometrial histology after 3 months of intravaginaladministration of the hormone precursor DHEA as shown byhistopathological examination of the endometrial biopsies obtainedbefore and after 12 weeks of treatment. These findings are in agreementwith the absence of aromatase activity in the human endometrium(Baxendale. Reed et al. 1981; Bulun, Lin et al, 2005), These findingsare also strongly supported by the well recognized clinical observationthat endometrial atrophy is characteristic of postmenopause despite thecontinuous secretion of DHEA thorought life (Labrie, Luu-The et al.2005; Labrie, Bélanger et al, 2006). The absence in the humanendometrium of the steroidogenic enzymes necessary to transform DHEAinto estrogens is in agreement with the physiological role of theendometrium which is active exclusively during the reproductive yearswhen its function is essentially controlled by hormones of ovarian andplacental origins. There is no physiological role of the endometriumafter menopause which would justify any continued action of estrogensafter cessation of estrogen secretion by the ovaries. Accordingly, theenzymes required for the synthesis of estrogens from DHEA are notexpressed in the endometrium which a tissue fully dependent uponestrogens of ovarian origin.

Estrogens administered alone have long been known to stimulateendometrial proliferation (Smith, Prentice et al. 1975) while progestinsadministered in combination with estrogens inhibit the stimulatoryeffect of estrogens (Feeley and Wells 2001). Since androgen receptorsare expressed in the human endometrium and stroma (Mertens, Heineman etal. 19%), it is of interest to mention that a clinical study whichinvestigated the effect of androgens showed no effect on the endometriumof a relatively high dose of testosterone in postmenopausal women(testosterone undecanoate, 40 mg every second day) (Zang, Sahlin et al,2007). In women who received estradiol valerate (2 mg/day), Ki labelingincreased by 50% at 3 months of treatment while simultaneousadministration of testosterone decreased proliferation to 28%. Ki67labeling was increased only in the two groups receiving estrogen but itwas decreased by the addition of testosterone in the stroma. Whilehaving no stimulatory effect on endometrial proliferation in women,testosterone appears to exert some antiestrogenic effect in theendometrium.

While the FDA guidance encourages sponsors to develop the lowest dosesand exposures for both estrogens and progestins, we must recognize thatalthough estrogens are efficient in correcting the symptoms of vaginalatrophy and vasomotor symptoms, systemic estrogens are not thephysiological hormones that permit 25% of postmenopausal women to avoidthe moderate to severe symptoms of vaginal atrophy. These women remainrelatively asymptomatic thorough all their postmenopausal years Sincethe only source of sex steroids in postmenopausal women, bothsymptomatic and asymptomatic, is local estrogen and androgenbiosynthesis from adrenal DHEA, by the mechanisms of intracrinology.Replacement with DHEA is the only physiological approach which permitsto provide women suffering from postmenopausal symptoms the missingamount of DHEA responsible for their symptoms. With the approach calledhormone precursor replacement therapy (HPRT), vaginal atrophy andvasomotor symptoms should be corrected with no more risk than that ofthe fellow postmenopausal women who have no symptoms of vaginal atrophybecause of a higher exposure to DHEA and the sex steroids madeintracellularly by the process of intracrinology.

Sex steroid precursors administered in accordance with the invention arepreferably administered in a dosage range (1) between 0.5 to 100 mg perday, (preferably 3 to 50 mg per day, and most preferably between 3 and13 mg per day), when intravaginally administered; (2) in a dosage rangebetween 15 to 200 mg per day (preferably 30 mg to 100 mg per day), whenadministered on the skin; (3) in a dosage range between 10 to 200 mg perday (preferably 25 mg to 100 mg per day), e.g., 75 mg per day, whenorally administered; or (4) in a dosage range between 1.0 to 25 mg perday (preferably 3.25 to 20 mg per day), when parentally administered(i.e. intramuscular, or subcutaneous).

In a pharmaceutical composition for vaginal administration, DHEA orother precursor is preferably present in a concentration between 0.1 and10% by weight relative to total weight of the composition morepreferably between 0.2 and 3.0 percent, especially between 0.25 and 2.0percent. For example, a 1.3 milliliter (mL) vaginal suppository having a0.5% DHEA (by weight of total composition), administered once daily,desirably provides 6.5 mg/day of DHEA. Larger or smaller suppositoriesmay be used, as may different concentrations, while maintaining dosagein the desired range.

In a pharmaceutical composition for administration on skin, DHEA orother precursor is preferably present in a concentration between 0.1 and10% by weight relative to total weight of the composition morepreferably between 0.2 and 2.0 percent, especially between 0.3 and 1.5percent.

In a pharmaceutical composition for oral administration, DHEA or otherprecursor is preferably present in a concentration between 5 and 98% byweight relative to total weight of the composition more preferablybetween 10 and 50 percent, especially between 15 and 40 percent.

In a pharmaceutical composition for parental administration (i.e.intramuscular, or subcutaneous), DHEA or other precursor is preferablypresent in a concentration between 0.2 mg/mL and 25 mg/mL, morepreferably between 0.65 aid 15 mg/mL especially between 2 mg/mL and 10mg/mL.

EXAMPLE OF EFFICAY OF THE INVENTION Example 1 Clinical Trail ERC-213DHEA Bioavailability Following Administration of Vaginal Suppositoriesin Post Menopausal Women with Vaginal Atrophy Phase I Randomized,Placebo-Controlled Pharmacokinetics and Local Action of DailyAdministration of DHEA Suppositories for One Week

The primary objective of that study was the evaluation of the systemicbioavailability of DHEA and its metabolites following daily intravaginalapplication of suppositories at four different DHEA concentrations. Thisstudy was a randomized, placebo-controlled and double-blind trial of10-subjects per arm. Forty postmenopausal women were thus randomized toreceive a daily dose of one suppository of the following DHEAconcentrations: 0.0%, 0.5% (6.5 mg of DHEA/suppository), 1.0% (13 mg ofDHEA/suppository) or 1.8% (23.4 mg of DHEA/suppository).

The maturation index as well as the vaginal pH were measured atpretreatment as well as after 7 days of treatment in order to obtain anindication of the local effect of DHEA during that short time period.

As illustrated in FIG. 1, Table 1 and Table 2, daily intravaginalapplication of a 1.3 ml suppository containing 0.5%. 1.0% and 1.8% DHEAled to a progressive increase of serum DHEA with AUC_(0-24 h) values of24.8±4.8 ng·h/ml, 56.2±8.9 ng·h/ml (p<0.05), 76.2±103 ng·h/ml (p<0.01)and 114.3±9.97 ng·h/ml (p<0.01), respectively. There was thus 127%, 207%and 361% increases over control at the 0.5%, 1.0% and 1.8% doses ofDHEA, respectively. As observed for all other steroids, similar valuesof the AUC_(0-24 h) were observed on days 1 and 7.

In fact, the average serum value of 4.76±0.42 ng/ml of DHEA followingtreatment with the highest dose (Table 2) is similar to the value of4.47±2.19 ng/ml found in forty-seven (47) 30-35 year-old premenopausalnormal women (Labrie, Banger et al, 2006). That serum DHEA following anyof the doses of DHEA used remains within the limits of normalpremenopausal women is well illustrated in FIG. 7A.

As observed previously following oral or percutaneous administration ofDHEA (Labrie, Bélanger et al, 2007), serum 5-diol follows a patternalmost superimposable to that of DHEA, although much lowerconcentrations are seen. In fact, the AUC_(0-24 h) value goes from5.60±0.60 ng·h/m in the placebo group on day 7 to 9.83±1.14 (p<0.05),13.8±1.87 (p<0.01) and 21.0±1.66 (p<0.01) at the 0.5%, 1.0% and 1.8%DHEA doses, respectively (1, Table 1). Such changes correspond to 75%,147% and 276% increases over control. Only the 1.8% DHEA dose causesincreases in serum 5-diol exceeding the values found in normalpremenopausal women (FIG. 7B) during the 24 h following dailyintravaginal administration of DHEA on day 7.

The AUC 0-24 h value of serum Testo showed no significant change at the0.5% dose (2.79±0.30 ng·h/ml versus 258±0.33 ng·h/ml in the placebogroup) (FIG. 2B). At the 1.0% and 1.8% doses, AUC 0-24 h values of4.54±0.91 ng·h/ml (p<0.05) and 5.97±0.69 ng·h/ml (p<0.01) were found(Table 1). These values translate into average serum Testo levels of0.11±0.01 (N.S.), 0.12±0.01 (N.S.), 0.19±0.04 (p<0.05) and 0.25±0.03(p<0.01) ng/ml, respectively. Even at the highest 1.8% DHEA dose used,serum Testo levels remained within the normal range of premenopausalwomen measured at 0.18±0.07 ng/m (0.06-0.31, 5th-95th centiles) (Labrie,Bélanger et al. 2006) (FIG. 7E). The 1.0% dose (0.18±0.07 ng/ml), on theother hand, corresponds exactly to the values found in normalpremenopausal women, namely 0.19±0.4 (FIG. 7E).

FIGS. 2C and D, serum DHT increased from an AUC_(0-24 h) value of0.58±0.07 ng·h/ml in the placebo group on day 7 to 0.93±0.11 (N.S.),1.31±0.26 (p<0.05) and 1.93±0.23 (p<0.01) ng·h/ml in the 0.5%, 1.0% and1.8% DHEA groups, respectively (Table 2). These values correspond toaverage serum DHT levels of 0.02±0.01, 0.04±0.01, 0.05±0.01 and0.08±0.01 ng/mil (Table 2), thus reaching, at the highest DHEA dose, thenormal serum DHT levels of 0,07±0.03 ng/ml observed in premenopausalwomen (Labrie, Bélanger et al. 2006) (FIG. 7F).

The average serum Et levels were measured at 12.6±1.41 ng/ml in theplacebo group on day 7 (Table 2) while there was no significant changeat the 0.5% DHEA dose (15.4±2.04 ng/ml). An increase to 24.1±3.54 ng/ml(p<0.01) and 25.0±2.85 ng/ml (p<0.01) was observed at the 1.0% and 1.5%DHEA doses, respectively. The corresponding AUC_(0-24 h) values areillustrated in FIG. 36 and are indicated in Table 1.

Average serum E₂ levels were measured at 2.77±0.29 pg/ml and 4.04±0.69pg/ml (N.S.) in the placebo and 0.5% DHEA groups, respectively (Table2). Average serum E₂ concentrations of 6.01±1.31 pg/ml (p<0.05) and5.68±0.84 pg/ml (p<0.05) were found on day 7 in women who received the1.0% and 1.8% DHEA doses for absolute increases of 3.18 and 2.85 μg/mover placebo, respectively. Comparable findings were observed for serumE₁-S with average serum levels of 0.12±0.02 ng/ml and 0.13 ng/ml (N.S.)in the placebo and 0.5% DHEA groups, respectively (Table 2). Values of0.18±0.03 ng/ml and 0.25±0.25 ng/ml were measured in the 1.0% and 1.8%DHEA groups, respectively. Only the 1.8% DHEA group shows a statisticaldifference (p<0.01) with the placebo group,

As can be seen in 4B and D, a comparable pattern is seen for both E₁-Sand DHEA-S. The AUC 0-24 value of serum DHEA-S was measured at 8.35±2.22ng·h/m in the placebo group and 13.3±3.16 ng·h/ml in the 0.5% DHEA group(N.S.). With the two higher DHEA doses, the AUC_(0-24 h) values weremeasured at 16.5±2.71 ng·h/m (N.S.) and 19.3±3.59 ng·h/ml (p<0.05),respectively (FIG. 4D, Table 1). These values of DHEA-S at all doses ofDHEA remain below the serum DHEA-S levels observed in premenopausalwomen which show an average of 1.27±0.62 ng/ml (7C).

As illustrated in FIG. 58, the AUC_(0-24 h) values of serum 4-dionefollowing DHEA administration on day 7 were measured at 6.34±0.80 and8.71±0.84 ng·h/ml (N.S.) in the placebo and 0.5% DHEA groups,respectively. At the two higher DHEA doses, the AUC a values of 4-dioneincreased slightly to 11.1±1.51 (p<0.01) and 11.9±0.81 (p<0.01) ng·h/ml,respectively. As can be seen in 7D and Table 2, all these values ofserum 4-dione remained well below the average serum 4-dineconcentrations observed in normal premenopausal women. In fact, thehighest DHEA dose led to average serum 4-dione concentrations of0.50±0.03 ng/ml while the average value in 30-35 year old cycling womenis 0.96±0.35 ng/ml (Labrie, Bélanger et al. 2006) (Appendix 2), thusreaching only 50% of the serum 4-dione levels observed in premenopausalwomen.

Considering the crucial role of measurements of the serum levels ofADT-G, 3α-diol-3G and 3α-diol-17G (Labrie, Bélanger et al. 2006) it isof interest to see in 5D and Table 2 that serum levels of ADT-Cincreased from an average value of 6.97±1.20 ng/ml in the placebo groupto 19.2±3.99 ng·h/ml in the 0.5% DHEA group (p<0.01). Values of19.7±2.48 and 25.7±2.88 ng·h/ml were measured in the 1.0% and 1.8% DHEAgroups, respectively (p<0.01 vs placebo for both DHEA-treated groups).Similar changes can be seen for the minor androgen metabolites3α-diol-3G and 3α-diol-17G (6B, 6D, 8 and 8C, Table 1 and Table 2). Itis important to indicate, as illustrated in FIG. 8, that even at thehighest dose of DHEA used, the average serum levels of ADT-G 3α-diol-3Gand 3α-diol-17G remained 36%, 11% and 6% below the average serum levelsfound in premenopausal women.

As shown in Table 2, the sum of the androgen metabolite glucuronidesmeasured over a 24 h period on day 7 of the administration of a 1.3 mlsuppository containing 1.8% DHEA (23.4 mg DHEA) is only 28.2 ng/ml whilethe mean serum concentration of the same metabolite in 30-35 year-oldpremenopausal women is 42.8 ng/ml (Labrie, Bélanger et al. 2006)(Appendix 2). Accordingly, the highest DHEA dose used leads to only65.7% of the value corresponding to the total androgen metabolites foundin normal cycling young women. The 1.5% and 1.0% DHEA doses, on theother hand, lead to sums of androgen metabolites of 21.02 ng and 21.53ng/ml, respectively, thus corresponding to only 49.0% and 50.2% of thevalues observed in premenopausal women (FIG. 9). We have previouslyfound that daily oral administration of 100 mg of DHEA leads to 74% ofthe levels found in premenopausal women (Labrie, langer et al, 2007).

We have previously observed that following oral or percutaneousadministration of DHEA, the changes in serum DHEA are an approximately100% overestimate of the changes in steroid formation reflected bychanges in serum ADT-G, 3α-diol-3G and 3α-diol-17G (Labrie, Banger etal. 2007). As can be seen in FIG. 9, average serum DHEA levels went from23% of the value observed in premenopausal women of the placebo group to52%, 71% and 106% in women who received the 0.5%, 1.0% and 1.8% DHEAdoses, respectively. The data of FIG. 9 indicate that changes in serumDHEA following intravaginal DHEA administration are also an overestimateof the changes in androgen formation and probably even more in estrogenformation as illustrated by the even smaller changes in serum E₁-S(Table 1). In fact, at the 1.0% dose, serum androgen metabolitesincreased by 31.6% of the value found in premenopausal women while serumDHEA increased by 49.1% (55% overestimate). At the highest DHEA dose,serum androgen metabolites increased by 47.1% while serum DHEA increasedby 83.5% (77% overestimate).

TABLE 1 Areas Under the Curve (AUC_(0-24h)) Values DHEA and Eleven ofits Metabolites on Days 1 and 7 of Daily Administration of IntravaginalDHEA Suppositories to 40-75 Year-Old Postmenopausal Women with VaginalAtrophy. DHEA 5-DIOL TESTO DHT E1 E2 DAY 1 DAY 7 DAY 1 DAY 7 DAY 1 DAY 7DAY 1 DAY 7 DAY 1 DAY 7 DAY 1 DAY 7 GROUP VALUE ng h/ml ng h/ml ng h/mlng h/ml ng h/ml ng h/ml ng h/ml ng h/ml pg h/ml pg h/ml pg h/ml pg h/mlPLACEBO MEAN 24, 47 24, 82 5, 55 5, 80 2, 71

2,58

0, 61 0, 58 305, 58 301, 92 69, 51 66, 49 SEM 4.80 4.77

0.34 0.33 0.06 0.07 34.56 30.77  7.63

DHEA 0.5% MEAN 65, 49 56, 17 10, 91 9, 83 2, 79 2, 79 0, 91 0, 93 336,52 369, 69 87, 79 96, 93 SEM 7.00 8.04 1.03 1.34 0.29 0.33 0.

0.

37.96 48.86 11.34 16.46 DHEA 1.0% MEAN 74, 82 76, 22 12, 09 13, 84 3, 794, 54 1, 11 1, 31 418, 08 578, 59 101, 57 144, 34 SEM 8.71 10.28 1.861.87 0.75 0.91 0.23 0.26 70.91 84.90 22.97 31.47 DHEA 1.8% MEAN 123, 52114, 30 18, 98 21, 04 5, 13 5, 97 1, 62 1, 93 433, 74 600, 93 89, 76136, 28 SEM 0.43 0.90 1.06 1.05 0.72 0.89 0.39 0.23 37.88

20.27 E1-S DHEA-S 4-DIONE ADT-G 3α-DIOL-3G 3α-DIOL-17G DAY 1 DAY 7 DAY 1DAY 7 DAY 1 DAY 7 DAY 1 DAY 7 DAY 1 DAY 7 DAY 1 DAY 7 GROUP ng h/ml ngh/ml μg h/ml μg h/ml ng h/ml ng h/ml ng h/ml ng h/ml ng h/ml ng h/ml ngh/ml ng h/ml PLACEBO 3, 15 2, 93 6, 71 8, 35 6, 23 6, 34 176, 53 167, 3912, 00 12, 00 12, 53 12, 20 0.52 0.47 2.41 2.22 0.71 0.80  30.86 26.570.00 0.00 0.53 0.20 DHEA 0.5% 3, 19 3, 24 13, 59 13, 29 9, 03 6, 71 474,10 461, 15 16, 01 16, 73 24, 68 26, 74 0.90 0.83 3.42 3.16 0.96 0.84126.90 36.77 2.93 1.97 4.70 5.02 DHEA 1.0% 3, 14 4, 37 14, 42 16, 49 10,28 11, 06 417, 73 471, 54 17, 12 20, 14 20, 88 24, 94 0.44 0.86 3.972.71 1.36 1.51  86.09 50.54 2.28 2.26 4.42 4.75 DHEA 1.8% 4, 23 5, 9314, 99 19, 33 10, 61 11, 94 510, 77 617, 73 20, 36 26, 02 22, 00 32, 239.76 1.31 2.62 3.58 0.63 0.81  52.78 89.01 2.31 3.36 2.65 4.35 a: Datafrom one patient were excluded

indicates data missing or illegible when filed

TABLE 2 Average Serum Steriod Levels of DHEA and Eleven of itsMetabolites on Day 1 and 7 of Daily Administration of Intravaginal DHEASuppositories to 40-75 Year-Old Postmenopausal Women with VaginalAtrophy. The values were obtained by dividing the AUC 0-24 h valuesmeasured on days 1 and 7 by 24 thus yielding the average serumconcentration of each steroid over 24-h period. Serum steriodconcentrations measured in 30-35 year-old premenopausal women are addedas reference. DHEA 5-DIOL TESTO DHT E1 E2 DAY DAY DAY DAY DAY DAY DAYDAY DAY DAY DAY DAY 1 7 1 7 1 7 1 7 1 7 1 7 ng ng ng ng ng ng ng ng pgpg pg pg GROUP VALUE h/ml h/ml h/ml h/ml h/ml h/ml h/ml h/ml h/ml h/mlh/ml h/ml PLACEBO MEAN 1, 02 1, 03 0, 23 0, 23 1, 11

0, 11 0, 026 0, 024 12, 73 15, 58 2, 90 2, 77 SEM 0.20 0.20 0.02 0.020.01 0.01 0.003 0.003 1.44 1.41 0.32 0.29 DHEA 0.5% MEAN 2, 73 2, 34 0,45 0, 41 0, 12 0, 12 0, 038 0, 039 14, 02 15, 40 3, 66 4, 04 SEM 0.350.37 0.06 0.05 0.01 0.01 0.004 0.004 1.50 3.04

.67 0.49 DHEA 1.0% MEAN 3, 12 3, 18 0, 58 0, 58 0, 16 0, 19 0, 046 0,055 17, 42 24, 11 4, 23 6, 01 SEM 0.28 0.43 0.07 0.08 0.03 0.04 0.0

0.011 2.90 2.54 3.90 1.31 DHEA 1.8% MEAN 5, 15 4, 76 0, 79 0, 88 0, 210, 25 0, 068 0, 081 18, 07 25, 04 3, 74 5, 68 SEM 0.28 0.42 0.54 0.52

0.00 0.008 0.010 1.37 2.85 0.43 0.54 30-35 YEAR- MEAN 4, 47 0, 49 0, 180, 07 53, 96 82, 05 OLD

2.36 0.20 0.07 0.08 23.26 42.18 PREMENO-

4.14 0.44 0.17 9.07 49.47 71.38 PAUSAL

1.83-0.34 0.26-0.34 0.06-0.30 0.03-0.14 23.74-

.46 22.06-186.07 WOMEN (

) (

) (

) (

) (

) (

) (

) (

 

) E1-S DHEA-S 4-DIONE ADT-G 3α-DIOL-3G 3α-DIOL-17G DAY DAY DAY DAY DAYDAY DAY DAY DAY DAY DAY DAY 1 7 1 7 1 7 1 7 1 7 1 7 ng ng μg μg ng ng ngng ng ng ng ng GROUP VALUE h/ml h/ml h/ml h/ml h/ml h/ml h/ml h/ml h/mlh/ml h/ml h/ml PLACEBO MEAN 0, 13 0, 12 0, 36 0, 36 0, 26 0, 26 7,366,97 0,50 0,50 0,52 0,51 SEM 0.01 0.02

0.08 0.08 0.08 1.29 1.26 0.

0.

0.

DHEA 0.5% MEAN 0, 13 0, 13 0, 57 0, 55 0, 36 0, 36 19,75 19,21 0,67 0,701,03 1,11 SEM

0.14 0.1 0.04 0.05 0.28 0.88 0.08 0.08 0.20 0.23 DHEA 1.0% MEAN 0, 13 0,18 0, 60 0, 69 0, 43 0, 46 17,41 19,65 0,71 0,84 0,87 1,04 SEM

0.13 0.11 0.08 0.08 2.75 2.48 0.16 0.34 0.36 0.36 DHEA 1.8% MEAN 0, 180, 25 0, 62 0, 81 0, 44 1, 50 21,28 25,74 0,85 1,08 0,92 1,34 SEM

0.18

2.05 2.

30-35 YEAR- MEAN 1, 19 1, 27 0, 96 40, 21 1, 21 1, 43 OLD

0.25 28.31

PREMENO-

1.04 0.92 31.82 1.06

PAUSAL

WOMEN (

) (

) (

) (

) (

) (

) (

) (

 

)

indicates data missing or illegible when filed

(Labrie, Bélanger et al. 2006)

It should be mentioned, however, as shown in Table 3, that there was astrong tendency for lower pre-treatment values of many steroids in theplacebo group. This is related to particularly low values in the placebogroup for DHEA, DHEA-S, 4-dione, Testo, DHT, E₂, ADT-G and 3α-diol-17G,Since all the average serum steroid values observed after administrationof the 0.5% and 1.0% doses of DHEA remain within or well below thevalues found in normal premenopausal women, no attempt was made tocorrect this apparent bias. It is of interest to mention that theaverage 24 h serum levels of all steroids measured on day 7 of dailyadministration of a 0.5% DHEA suppository correspond almost exactly tothe values measured in normal 55- to 65-year-old women while the 1.0%DHEA suppository leads to values within the range observed in 55- to65-year old normal women (Labrie, Bélanger et al. 2006).

Since the androgen metablites are the most reliable measure oftransformation of exogeneous DHEA into active androgens, the presentdata indicate that even the highest dose of DHEA used in the presentstudy meet the FDA requirements of serum steroid levels which remainwithin the normal range found in normal premenopausal women.

TABLE 3 Basal Serum Steroid Levels on Days 1 and 7 of DailyAdministration of Intravaginal Increasing Doses of DHEA Data areExpressed in ng/ml Except for E₁ and E₂ (pg/ml) and DHEA-S (μg/ml).Steroid Placebo DHEA 0.5% DHEA 1.0% DHEA 1.8% DHEA Day 1 0.72 ± 0.141.09 ± 0.24 0.94 ± 0.19 0.99 ± 0.15 Day 7 0.69 ± 0.14 1.29 ± 0.26 1.43 ±0.19 1.83 ± 0.13 5-diol Day 1 0.22 ± 0.02 0.26 ± 0.03 0.26 ± 0.05 0.25 ±0.02 Day 7 0.22 ± 0.02 0.31 ± 0.05 0.36 ± 0.05 0.46 ± 0.04 DHEA- Day 10.372 ± 0.102 0.543 ± 0.157 0.572 ± 0.144 0.447 ± 0.094 S Day 7 0.368 ±0.100 0.592 ± 0.160 0.717 ± 0.125 0.805 ± 0.143 4-dione Day 1 0.18 ±0.02 0.21 ± 0.03 0.23 ± 0.04 0.22 ± 0.03 Day 7 0.16 ± 0.02 0.25 ± 0.030.34 ± 0.06 0.38 ± 0.03 Testo Day 1 0.10 ± 0.01 0.09 ± 0.01 0.12 ± 0.030.15 ± 0.03 Day 7 0.09 ± 0.01 0.10 ± 0.01 0.18 ± 0.03 0.23 ± 0.03 DHTDay 1 0.024 ± 0.003 0.026 ± 0.003 0.037 ± 0.010 0.029 ± 0.002 Day 70.023 ± 0.002 0.035 ± 0.004 0.047 ± 0.010 0.062 ± 0.006 E₁ Day 1 11.98 ±1.65  11.83 ± 1.28  14.72 ± 2.79  13.57 ± 1.88  Day 7 11.71 ± 1.19 13.53 ± 1.66  22.15 ± 3.21  23.77 ± 3.35  E₂ Day 1 3.00 ± 0.44 3.13 ±0.37 4.30 ± 1.38 3.42 ± 0.62 Day 7 2.75 ± 0.28 3.94 ± 0.65 5.98 ± 1.266.00 ± 1.10 E₁-S Day 1 0.137 ± 0.024 0.133 ± 0.029 0.117 ± 0.016 0.164 ±0.033 Day 7 0.143 ± 0.025 0.151 ± 0.034 0.203 ± 0.016 0.529 ± 0.049ADT-G Day 1 7.42 ± 1.48 13.65 ± 3.71  11.49 ± 2.10  9.44 ± 1.23 Day 76.72 ± 1.17 17.02 ± 4.39  16.34 ± 2.30  19.26 ± 1.96  3a-diol- Day 10.50 ^(a) 0.61 ± 0.07 0.71 ± 0.14 0.58 ± 0.05 3G Day 7 0.50 ^(a) 0.75 ±0.11 0.96 ± 0.25 0.96 ± 0.15 3a-diol- Day 1 0.50 ^(a) 0.84 ± 0.16 0.85 ±0.22 0.65 ± 0.06 17G Day 7 0.50 ^(a) 0.95 ± 0.16 1.18 ± 0.30 1.27 ± 0.19^(a) Steroid levels are below the limit of quantification for allsubjects (limit of quantification = 0.50 ng/mL).

After only one week of daily administration of the DHEA suppositories,the maturation index increased by 107% (p<0.01), 75% (p<0.05) and 150%(p<0,01) in the 0.5%, 1.0% and 1.8% DHEA groups, respectively (FIG.10A). No change was observed in the placebo group between day 1 and day7. Vaginal pH, on the other hand, decreased from 6.29±0.21 to 5.75±0.27(p<0.05), 6.47±0.23 to 5.76±0.22 (p<001) and 6.53±0.25 to 5.86±0.28(p<0.05), respectively in the 0.5%, 1.0% and 1.8% DHEA groups (FIG.10B). No change of vaginal pH was observed in the placebo group.

Example 2 Bioavailability and Metabolism of Oral and PercutaneousDehydroepiandrosterone in Postmenopausal Women 1. Introduction

Humans, along with the other primates, are unique among animal speciesin having adrenals that secrete large amounts of the inactive precursorsteroids DHEA and especially DHEA-S, which are converted into activeandrogens and/or estrogens in peripheral tissues (Labrie, 1991; Labrie,Banger et al., 1995; Labrie, Luu-The et al, 1997; Labrie, Simard et al,1996; Labrie, Luu-The et al., 2005; Labrie, Poulin et al., 2006 andSimpson 2000), In fact, plasma DHEA-S levels in adult men and women are100-500 times higher than those of testosterone and 1000-10,000 timeshigher than those of estradiol, thus providing a large reservoir ofsubstrate for conversion into androgens and/or estrogens in theperipheral intracrine tissues which possess the enzymatic machinerynecessary to transform DHEA into active sex steroids (Labrie 1991 andLabrie, Luu-The et al., 2005). In fact, the term intracrinology wasfirst coined in 1988 (Labrie, Bélanger et at, 1988) to describe thesynthesis of the active steroids made in the same cells where they exerttheir action with no or minimal release into the extracellular space andgeneral circulation before being inactivated (Labrie, 1991).

The marked reduction in the formation of DHEA-S by the adrenals duringaging (Banger et al., 1994; Vermeulen and Verdonck, 1976; and Migeon etal., 1957) results in a dramatic fall in the formation of androgens andestrogens in peripheral target tissues, a situation potentiallyassociated with age-related diseases such as insulin resistance(Schriock et al. 1988 and Coleman et al, 1982) and obesity (Nestler etal 1988; MacEwen and Kurman, 1991, and Tchernof et al. 1995).Moreover.much attention has been given to the benefits of DHEA administered topostmenopausal women, especially on the bone, skin, vaginum, glucose andinsulin metabolism, fat mass, as well as well-being after oral(Villareal and Holloszy, 2004; Baulieu et al., 2000; Morales, et al.1994; and Kawano et al. 2003) and percutaneous (Diamond et al., 19% andLabrie Diamond et al. 1997) administration. It thus becomes ofparticular importance to obtain more precise knowledge about thebioavailability, pharmacokinetics and metabolism of DHEA following thesetwo routes of administration.

Since we have already shown, using a pharmacological dose of DHEAadministered percutaneously for 2 weeks, that measurements of serumtestosterone (testo) and estradiol (E₂) levels do not provide a reliableassessment of the true intracellular pool of androgens and estrogens(Labrie, Bélanger et al., 1997; Labrie, Bélanger et al 2006 and Labrie,Bélanger, et al, 2007b) we have compared the serum levels of DHEA andnine steroids known to be most closely associated with active androgensand estrogens and their metabolites. A detailed analysis of the 24 hchanges of serum steroid levels was performed on the first day and after2 weeks of daily administration of DHEA by the oral route as well aspercutaneously using a DHEA cream or gel.

2. Subjects and Methods

Thirty-six healthy 60-70-year-old postmenopausal women participated inthe study after IRB approval and having given their written informedconsent. Body weight was within ±20% of normal body weight according toMetropolitan Life Tables.

No subject suffered from a significant metabolic or endocrine disorder,coronarian disease or hypertension. No women had treatment withandrogens or anabolic steroids within 6 months prior to the screeningvisit. All participants had a medical history, complete physicalexamination and serum biochemistry profile including lipids, completeblood count, urine analysis and detailed serum hormone determinationsduring the screening phase of the protocol.

3. Study Design, Treatment and Measurements

This study was a randomized open-label trial of 12 subjects per arm.After written informed consent was obtained and women were foundeligible, each subject was randomized to receive DHEA by cream, get ororally. Daily, before breakfast, for 14 days, subjects received, at theresearch clinic, either 4 g of 10% DHEA gel or 4 g of 10% DHEA creamapplied on a total 30 cm×30 cm area of the thighs or two 50 mg capsulesof DHEA orally before breakfast.

Blood sampling was performed at 08:00-09:00 h at screening and beforeapplication of DHEA, on the first day of dosing, as well as on days 2,4, 7, 10 and 14. On the 1st and 14th days, blood samples were obtainedat 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 12 h and 24 hfollowing DHEA administration. 4. Serum steroid analysis

DHEA, DHEA-S, androst-5-ene-3β,17β-diol (5-diol), testosterone,androstenedione (4-dione), 17β-estradiol (E₂), estrone (E₁), estronesulfate (E₁-S), androsterone glucuronide (ADT-G), andandrostane-3,17β-diol glucuronide (3 α-diol-G) were measured by gaschromatography/mass spectrometry (DHEA, 5-diol, 4-dione, testosterone,E₁ and E₂) using electron impact or chemical ionization and by liquidchromatography/tandem mass spectrometry using turboionspray (DHEA-S,E₁-S, ADT-G and 3α-diol-G) as described (Labrie, Bélanger et a., 2006;Labrie, Bélanger, et al, 2007b and Swanson et al. 2007).

5. Calculations and Statistical Analysis

On days 1 and 14, the area under the curve of the serum concentration ofeach steroid was measured between 0 h and 24 h (AUC_(0-24 h)). The areasunder the curves were calculated by a linear trapezoidal method(model-independent). The relative bioavailability of the DHEA gel, DHEAcream and DHEA capsules was based on the mean difference in thelog-transformed AUC values, All calculations were performed with the SASsoftware (SAS Institute, Cary, N.C., USA).

6. Results

The oral administration of two capsules of 50 mg of DHEA led to anincrease of serum DHEA from 2.3±0.3 ng/ml to a maximal value of 15.6±2.5ng/ml at 1 h with a progressive decrease thereafter to 5.7±0.5 ng/ml at6 h followed by a plateau up to 24 h (FIG. 11A). When 4 g of a 10% DHEAgel or cream were applied on a 30 cm×30 cm area of the skin of thethighs, serum DHEA levels only started to increase at 1.2 h to reachvalues of 8.2±2.0 and 8.0±1.2 nmol/l, respectively, at 24 h (FIG. 11A).There was no significant difference between the cream or gel in theserum levels of DHEA at any of the time intervals studied up to 24 hafter first application of the precursor steroid on the skin.

When serum 5-diol was measured after oral first administration of DHEA,the concentration of 5-diol increased from a pretreatment concentrationof 0.31±0.03 ng/ml to a maximal value of 1.19±0.13 ng/ml at 1 h with aslow and progressive decrease thereafter to reach 0.79±0.05 ng/ml at 24h (FIG. 11B). It can be seen in the same figure that the serum levels of5-diol increased much more slowly after administration of DHEApercutaneously by cream or gel to reach the first statisticallysignificant different values of 1.00±0.14 ng/ml for the cream and0.72±0.14 ng/ml for the gel at 24 h.

Following oral DHEA, serum 4-dione increased from 0.6±0.1 ng/ml to amaximal value of 9.5±2.2 ng/ml at 1 h followed by a rapid decreasethereafter to values which remained on a plateau of about 1.2 ng/mlbetween 8 h and 24 h (FIG. 12A). Following administration of DHEA bycream or gel, on the other hand, the first significant increase of serum4-dione was only observed at 24 h at values of 0.9±01 and 0.8±0.1 ng/mlfor the cream and gel, respectively.

A comparable pattern was observed for serum testosterone. In fact, afteroral administration of two 50 mg capsules of DHEA, serum testosteroneincreased from 0.38±10.03 ng/ml to a maximal value of 0.79±0.14 ng/milat I h. This rise was followed by a rapid decrease to 0.30±0.08 ng/ml at6 h followed by a plateau thereafter until 24 h (FIG. 12B). When DHEAwas applied as cream or gel, the first increase was observed at 24 h ata value of approximately 0.45 ng/ml. As can be seen in FIGS. 13A and B,the first administration of DHEA by the oral or percutaneous route hadno statistically significant effect on the serum levels of E₁ or E₂during the first 24 h.

Serum DHEA-S on the other hand followed a pattern similar, althoughslightly delayed, compared to DHEA and 5-diol following oraladministration of two capsules of 50 mg DHEA (FIG. 14A). Thus, serumDHEA-S increased from 0.4±10.1 μg/ml to 7.7±1.0 μg/ml at 1 h to amaximal value of 8.4±10.6 μg/ml at 2 h with a progressive decrease to2.7±0.3 μg/ml at 24 h. No significant change of serum DHEA-S wasobserved during the first 24 h after administration of DHEA in a creamor gel. Serum E₁-S, on the other hand, did not change significantlyduring the first 24 h following the first

Serum ADT-G, the main metabolite of androgens, increased from 14±3 ng/mlto 760±150 ng/ml at 1 h and 790±140 ng/ml at 2 h to then decreaseprogressively to 92±5 ng/ml at 12 h and 70±5 ng/ml at 24 h (FIG. 15A).Serum 3 α-diol-G, on the other hand, increased from 2.2±0.5 ng/ml to14.5*2.0 ng/ml at 2 h (FIG. 15B). The decrease observed thereafter for 3α-diol-G was however much slower than that of ADT-G, a decrease of onlyabout 40% being observed between 2 h and 24 h after oral administrationof DHEA. Following application of 4 g of 10% DHEA on the skin, there wasno significant change of serum ADT-G or 3 ca-diol-G up to 24 h (FIG.15B).

When the measurements of the same kinetic parameters were repeated onthe 14th day of daily dosing, it could be seen that the administrationof two capsules of 50 mg of DHEA led, from a predosing value of 4.2±0.4ng/ml, to a maximal concentration of 14.8±4.4 ng DHEA/ml at 1 h followedby a progressive decrease thereafter to 4.5±0.4 ng/ml at 24 h (FIG.16A). On the other hand, when DHEA was administered by cream or gel, nosignificant change was observed during the 24-h period and serum DHEAremained between 10 ng/ml and 15 ng/m following application of the creamand between 7 ng/ml and 11 ng/ml following application of the gel.

Similarly, when serum 5-diol was measured on the 14th day of treatment,the serum concentration of this steroid increased from 0.46±0.04 ng/mlto 1.37±0.21 ng/ml at 1 h with a slow decrease thereafter to reach0.64*0.06 ng/ml at 24 h (FIG. 161). As observed for DHEA, serum 5-diolremained approximately constant during the 24-h period at about 1.5-1.9ng/ml following application of the cream and 1.0-1.3 ng/ml followingapplication of the gel.

When serum 4-dione was measured on the 14th day of dosing, the serumconcentration of this steroid increased from 1.3±0.2 ng/ml to a maximalvalue of 9.8±1.7 ng/ml at 1 h followed by a rapid decrease to 1.5±0.1ng/ml at 6 h with a value of 1.2±0.1 ng/ml measured at 24 h (FIG. 17A).Following application of DHEA on the skin as a cream or gel, there was anon-significant increase of serum 4-dione to approximately 2.5 ng/ml at2 h with values, thereafter, remaining on a plateau at 1.0-1.6 ng/ml upto 24 h (FIG. 17A).

Serum testosterone increased on the 14th day of dosing following oraladministration of 100 mg of DHEA from 0.31±0.04 ng/ml to a maximal valueof 0.83±0.11 ng/ml at 1 h followed by a progressive decrease to a valueof 0.37±0.04 ng/ml at 24 h (FIG. 17B).Following DHEA application as acream or a gel, serum levels of testosterone remained unchanged duringthe 24-h period at approximately 0.3 ng/ml, this value being notsignificantly different from pretreatment. As observed on the first day,there was no significant change in the serum levels of E1 (FIG. 18A) orE2 (FIG. 18B) during the 24 h which followed the 14th dailyadministration of DHEA by the oral or percutaneous route.

From a predosing level of 1.95±0.15 μg/ml, serum DHEA-S increased to8.3±0.4 μg/ml at 1 h to decrease progressively to 2.6±0.3 μg/ml at 24 h(FIG. 19A). No significant change in serum DHEA-S was observed afterapplication of DHEA on the skin. Serum E-S, on the other hand, did notchange during the 24 h following the 14th daily administration of DHEAby the oral or percutaneous route (FIG. 19B).

While starting at a higher level on day 14 than on day 1, serum ADT-Gincreased rapidly from 66±1 ng/m to 996±105 ng/l at 1 h to decreaseprogressively thereafter to 116 ng/ml at 12 h and 91±15 ng/ml at 24 h(FIG. 20A). No significant change in serum ADT-G levels occurredfollowing the application of DHEA on the skin. Serum 3 α-diol-G, on theother hand, increased from 12±2.5 ng/ml to 29.4±5.5 ng/ml at 2 h todecrease slowly thereafter to reach 13±3.0 ng/ml at 24 h following 14thdaily oral administration of 100 mg DHEA. No significant change wasobserved on serum 3 α-diol-G after percutaneous administration of DHEA(FIG. 20B).

In order to obtain a more precise measure of the accumulation of DHEAand its metabolites, we next compared the areas under the curves of theserum steroid concentrations (AUC_(0-24 h) values) measured on the 1stand 14th days of dosing. As can be predicted from FIG. 11, FIG. 12, FIG.13, FIG. 14, FIG. 15, FIG. 16, FIG. 17, FIG. 18, FIG. 19, and FIG. 20the AUC_(0-24 h) values of all steroids, except the metabolites ofestrogens (E₁-S) and androgens (ADT-G and 3 α-diol-G), due to someaccumulation of these steroids, are similar on the 1st and 14th days ofadministration of DHEA by the oral route (Table 1). Followingpercutaneous administration of DHEA, on the other hand, due to theslower absorption of DHEA following administration in a cream or gel,155% and 86% higher values of the DHEA AUC_(0-24 h) values are observedon the 14th day compared to the first day of dosing, respectively,Higher values are also observed for all the other steroids, except forE₁, E₂ and testosterone which showed no Table 4.

TABLE 4 AUC_(0-24 h) values measured on the 1st and 14th days of dosingas well as their ratio DHEA 5-Diol 4-Dione Testosterone E1 E2 DHEA-SE1-S ADT-G 3α-Diol-G Steriod (ng h/ml) (ng h/ml) (ng h/ml) (ng h/ml) (pgh/ml) (pg h/ml) (pg h/ml) (pg h/ml) (ng h/ml) (ng h/ml) 2 × 50 mgcapsules 1st dosing 153 (19) 19.0 (20) 40.2 (39) 9.47 (31) 745 (30) 136(20) 106 (20) 4.81 (39) 4112 (24) 259 (44) 14th dosing 144 (26) 20.4(25) 43.6 (27) 9.72 (23) 910 (23) 165 (25) 95.0 (16) 7.44 (36) 5607 (28)453 (41) 14th/1st 0.94 1.07 1.08 1.03 1.22 1.21 0.88 1.55 1.36 1.75 4 g10% cream 1st dosing 107 (33) 13.7 (31) 12.3 (43) 8.35 (16) 680 (48) 147(50) 10.7 (46) 4.83 (58) 40.4 (62) 50.6 (93) 14th dosing 273 (36) 39.7(31) 22.8 (33) 8.77 (16) 847 (22) 173 (27) 199 (34) 7.96 (39) 977 (66)114 (104) 14th/1st 2.55 2.91 1.85 1.00 1.24 1.19 1.86 1.65 2.42 2.25 4 g10% gel 1st dosing 101 (49) 10.3 (55) 13.3 (45) 8.76 (11) 620 (31) 214(137) 11.2 (35) 5.53 (64) 254 (30) 38.3 (86) 14th dosi 188 (30) 27.2(32) 21.3 (51) 8.04 (22) 785 (40) 152 (24) 18.6 (34) 9.11 (106) 455 (23)60.3 (85) 14th/1st 1.86 2.64 1.60 0.96 1.27 0.71 1.66 1.65 1.79 1.57Values within parenthesis represent % coefficient of variation. DHEA wasadministered by the oral route (2 × 50 mg capsules) or followingapplication on the skin of 4 g of 10% DHEA cream or 4 g of 10% gel.

As can be clearly seen in Table 5 and FIG. 21 there was no significantchange in the serum E₁, E₂ or testosterone AUC_(0-24 h) values measuredon the 14th day of dosing compared to the predosing levels. Significantincreases, however, were observed for all two other steroids. Thus,following daily oral dosing with 100 mg DHEA for 2 weeks, the area underthe concentration curve of DHEA measured during the 24 h followingadministration of the steroid increased 167% over the pretreatment valuewhile for 5-diol, 4-dione, DHEA-S, E-S, ADT-CG and 3 α-diol-C,respective increases of 138%, 238%, 873%, 60%, 1820% and 874% wereobserved.

TABLE 5 Pretreatment and 14th day AUC_(0-24 h) values of DHEA and itsmetabolites Testos- 3□□- DHEA 5-Diol 4-Dione terone E1 E2 DHEA-S E1-SADT-G Diol-G Steriod (ng h/ml) (ng h/ml) (ng h/ml) (ng h/ml) (pg h/ml)(pg h/ml) (pg h/ml) (pg h/ml) (ng h/ml) (ng h/ml) Basal 53.9 8.56 12.96.72 717 135 9.76 4.64 292 46.6 (pretreatment) (A) 2 × 50 mg capsules14th day 1.44 20.4 43.6 9.72 910 165 95.6 7.44 5607 453 14th/basal 2.672.38 3.38 1.11 1.27 1.22 9.73 1.60 19.2 9.74 (B) 4 g 10% cream 14th day2.73 39.7 22.8 8.77 847 175 19.9 7.96 977 114 14th/basal 5.06 4.63 1.771.01 1.18 1.30 2.04 1.72 3.34 2.45 (C) 4 g 10% gel 14th day 188 27.221.3 8.04 785 152 18.6 9.11 455 60.3 14th/basal 3.49 3.18 1.65 092 1.091.13 1.91 1.96 1.56 1.30 DHEA was administered by the oral route orpercutaneously by cream or gel. Basal AUC_(0-24 h) values werecalculated by multiplying the pretreatment basal serum steroid levelsincluding screening by 24 h.

Except for DHEA and 5-diol, lower increases were observed followingadministration of DHEA cream or gel, In fact, following application ofthe DHEA cream, the DHEA-S AUC_(0-24 h) value increased by only 104%while the 4-dione, E₁-S, ADT-G and 3α-diol-G AUC_(0-24 h) valuesincreased by 77%, 72%, 234% and 145% over control, respectively. The AUCvalues for DHEA and 5-diol, on the other hand, increased by 406% and363%, respectively (Table 5, FIG. 21). Comparable but somewhat lowerincreases were observed with the DHEA gel where the serum 4-dione,DHEA-S, E₁-S, ADT-G and 3 α-diol-GAUC_(0-24 h) values increased by 65%,91%, 96%, 56% and 30% over control while the AUC_(0-24 h) values forDHEA and 5-diol increased by 249% and 238%, respectively.

Our recent findings (Labrie, Bélanger et al., 2007b) have shown that theserum DHEA changes observed following exogenous DHEA administration areat least a 100% overestimate of the true changes in sex steroidformation. In support of these data, FIG. 21 shows that following DHEAadministration by cream or gel, the changes in serum DHEA are a markedoverestimate of the changes in serum levels of all the steroids measuredexcept for 5-diol, the immediate metabolite of DHEA. For the DHEA cream,the changes in the AUC_(0-24 h) values of serum 4-dione, DHEA-S, E-S,ADT-G and 3 α-diol-G are only 77%, 14%, 72%, 234% and 145% compared tothe 406% increase over pretreatment levels observed for serum DHEA.

For the androgens, it is now well established that uridine glucuronosyltransferase 267 (UGT 287), UGT 2815 and UGT 2817 are the three enzymesresponsible for the glucuronidation of all androgens and theirmetabolites in the human (Bélanger et al. 213). This recent completionof the identification and characterization of all the humanUDT-glucuronosyl transferases makes possible the use of the glucuronidederivatives of androgens as markers of total androgenic activity in bothwomen and men (Labrie, Bélanger et al., 2006; Labrie, Bélanger, et al,2007b and Swanson et al. 2007). Accordingly, since all androgens aremetabolized into ADT- and 3 α-diol-G, the estimate of the percentage ofefficacy of percutaneous DH EA for transformation into active androgensis thus estimated at 52% when adding the changes in ADT-G and 3 α-δtoλ-Γ(a weighted 211% value compared to the DHEA changes of 406%). Similarly,following DHEA gel administration, the 249% increase in serum DHEAtranslates into only 65%, 91%, 96%, 56% and 30% increases in theAUC_(0-24 h) values of serum 4-dione, DHEA-S, E1-S1, ADT-G and 3α-diol-G, respectively.

Since the high level of glucuronidation in the intestine and liverexplains the high serum level of ADT-G and 3 α-diol-G (Bélanger et al.2003) following oral administration of DHEA, the relatively smallincrease in serum E₁-S (60%) compared to the 167% increase in serum DHEAafter oral DHEA indicates a 36% relative efficacy of transformation intoestrogens. As shown earlier (Labrie, Bélanger et al., 1997; Labrie,Banger et al., 2006; Labrie, Bélanger, et al, 2007b), the present dataindicate that DHEA administrated to postmenopausal women ispredominantly transformed into androgens rather than into estrogens.

Discussion

The present data clearly show that during chronic treatment with DHEA bycream or gel, the concentration of all the steroids rapidly reaches aplateau with no detectable change in the serum concentration of any ofthe steroids measured during daily application of DHEA on the skin.Accordingly, from 24 h after first administration of DHEApercutaneously, the concentration of all steroids remains at the samelevel, thus showing that daily application of DHEA on the skin maintainsconstant serum levels of DHEA and all its metabolites. In postmenopausalwomen, it is already known that the circadian variation of serum DIEA isrelatively small compared to the situation in normally cyclingpremenopausal women (Lui and Laughlin, 1990).

The present data also show that following daily oral administration ofDHEA, there is no significant accumulation of DHEA or of itsmetabolites. Moreover, the metabolism of DHEA following itsadministration by the oral or percutaneous route is quantitativelysimilar, the quantitative differences being explained by theentero-hepatic metabolism following oral administration.

The higher AUC_(0-24 h) values of serum DHEA-S, ADT-G and 3 α-diol-Gcombined with the lower AUC_(0-24 h) values of DHEA and 5-diol followingoral versus percutaneous administration indicate that metabolism throughthe gastrointestinal tract and/or first passage through the liver leadsnot only to a higher level of transformation of DHEA into DHEA-S throughthe activity of DHEA-sulfotransferase (Luu-The et al., 1995) but also toan increased metabolism of DHEA into androgens and their inactivationthrough the activity of liver glucuronosyltransferases (Bélanger et al.2003; Turgeon et al., 2001 and Hum et al., 1999). n fact, as shown inTable L the exposure to DHEA of 144 ng h/m (AUC_(0-24 h)) on the 14thday of oral administration of 1) mg of DHEA leads to AUC_(0-24 h) valuesof 5607 ng h/ma and 453 ng h/ml for ADT-G and 3α-diol-G, respectively.On the other hand, after percutaneous administration with the 10% DHEAcream, the AUC values for DHEA, ADT-C and 3 α-diol-G are 273 ng h/ml,977 ng h/ml and 114 ng h/mi, respectively. Thus, after oraladministration, 1 ng h/m of DHEA corresponds to an AUC value of 42.1 ngh/ml for the combination of the two metabolites of androgens(ADT-G+3α-diol-G) while following application of the DHEA cream, 1 ngh/ml of DHEA exposure corresponds to 4.0 ng h/ml for the sum of the twoandrogen metabolites. Such data indicate that administration of DHEA bythe oral route leads to an approximately 10-fold higher level oftransformation of DHEA into ADT-G and 3 α-diol-G than after percutaneousadministration, at least at the doses used. When the same calculationsare made for the data obtained after administration of DHEA by gel, anexposure to DHEA of 1 ng h/ml is accompanied by an AUC_(0-24 h) value of2.7 ng h/ml for ADT-G+3 α-diol-G, thus indicating an even higher ratiobetween oral and percutaneous DHEA administration.

As shown in Table 4 while a DHEA AUC_(0-24 h) value of 1 ng h/ml leadsto an AUC_(0-24 h) value of 660 ng h/ml for DHEA-S following oraladministration of DHEA, corresponding values of 73 ng h/l and 99 ng h/lare observed after application of the precursor steroid by cream or gel.There is thus a 6.7-9.0-fold higher amount of DHEA-S in the circulationfollowing the same exposure to circulating DEA (serum AUC_(0-24 h)value) after oral compared to percutaneous administration of DHEA underthe conditions tested, The present data show a comparable influence ofthe passage of DHEA through the gastro-intestinal tract and the liver onserum DHEA-S, ADT-C and 3 α-diol-G levels.

Although a lower difference is seen, relatively higher levels of serum4-dione are observed after oral administration of DHEA compared topercutaneous administration of the precursor steroid. Thus, after oraladministration of DHEA, a 1 ng h/m value of the DHEA AUC_(0-24 h) leadsto a 0.3 ng h/m 4-dione AUC_(0-24 h) value while values of 0.08 ng h/mland 0.11 ng h/ml are observed after administration of DHEA by cream andgel, respectively. As measured in the circulation, the transformation ofDHEA into 4-dione is thus 2.70-3.76 times higher following oral comparedto percutaneous administration of DHEA.

The data of Table 5 show that the DHEA AUC_(0-24 h) value is increasedby 167% over control following the daily oral administration of 100 mgDHEA compared to pretreatment basal levels while the daily percutaneousadministration of 4 g of 10% DHEA cream and gel increases the serum DHEAlevels by 406% and 249%, respectively. Since 4(mg of DHEA were appliedon the skin compared to 100 mg by the oral route, and assuminglinearity, the present data indicate that the oral route is 2.9- and48-fold more efficient compared to the formulation used for the DHEAcream and gel, respectively.

In a study also performed in postmenopausal women, the oraladministration of 150 mg and 300 mg of micronized DHEA resulted inmaximal serum DHEA-S, DHEA and testosterone of approximately 1.5 mg/ml,15 ng/ml and 2.75 ng/ml after the 300 mg dose and 10 μg/ml, 12 μg/ml and1.6 ng/ml after the 150 mg DHEA dose, respectively (Buster et al. 1992).Examination of these early results shows that a 20-fold increase inserum DHEA-S led to only a 6.9-fold increase in serum testosterone whileserum DHEA was increased to 11.6-fold. Moreover, when the measured serumtestosterone values are adjusted to one-third to take into account thetwo-thirds non-specific binding in the radioimmunoassay, the serumtestosterone levels remained within the physiological levels during the12 h which follow the administration of the 150 mg DHEA dose (Buster etal. 1992).

Similar differences observed between the oral and percutaneous routesfor serum DHEA are seen for 5-diol which is transformed directly fromDHEA by 171-hydroxysteroid dehydrogenase (Labrie. Luu-The, et al. 2000).In fact, while the 5-diol AUC_(0-24 h) value is increased byapproximately 138% over control after oral administration of 100 mg ofDHEA, increases of 363% and 218% are measured after application of 400mg of DHEA cream and gel. respectively.

As mentioned above, man is unique, with some other primates, in havingadrenals that secrete large amounts of the precursor steroids DHEA andDHEA-S, which are converted into 4-dione and then into potent androgensand/or estrogens in peripheral intracrine tissues (Labrie, 1991; Labrie,Belanger, et al, 1995; Labrie et al. 19; Labrie, Luu-The, et al, 1997;Simpson, 2000; Labrie, Luu-the, et al. 2005; Labrie, Poulin, et al.,2006 and Labrie, Bélanger, et al., 1998). It is thus remarkable thatman, in addition to possessing very sophisticated endocrine andparacrine systems, has largely vested in sex steroid formation inperipheral tissues Labrie, 1991) and (Bélanger, et al., 1998). In fact,while the ovaries and testes are the exclusive sources of androgens andestrogens in lower mammals, the situation is very different in man andhigher primates, where active sex steroids are in large part or whollysynthesized locally in peripheral tissues, thus providing target tissueswith controls which adjust the formation and metabolism of sex steroidsto local requirements.

Adrenal secretion of DHEA and DHEA-S increases during adrenarche inchildren at the age of 6-8 years, and maximal values of circulatingDHEA-S are reached between the ages of 20 and 30 years. Thereafter,serum DHEA and DHEA-S levels decrease markedly (Banger et al. 1994). Infact, at 70 years of age, serum DEA-S levels are decreased toapproximately 20% of their peak values, while they can decrease by 95%by the age of 85-90 years (Banger et al, 1994) and (Migeon et al.,1957). The 70-95% reduction in the formation of DHEA and DHEA-S by theadrenals during aging results in a dramatic reduction in the formationof androgens and estrogens in peripheral target tissues (Labrie,Bélanger et al., 2006). Such a marked decrease in the formation of sexsteroids in peripheral tissues could well be involved in thepathogenesis of a series diseases associated with aging.

As mentioned earlier, transformation of DHEA and DHEA-S into activeandrogens and/or estrogens in peripheral target tissues depends upon thelevel of expression of the various steroidogenic and metabolizingenzymes in each cell type (Labrie, 1991). Elucidation of the structureof most of the tissue-specific genes that encode the steroidogenicenzymes responsible for the transformation of DHEA and DHEA-S intoandrogens and/or estrogens has permitted rapid progress in this area(Labrie, Bélanger et al., 1995; Labrie, Lu-The et al., 1997; Labrie,Simard et al., 1996; Labrie, Luu-The et al., 2005; Labrie, Poulin etal., Labrie, Luu-The, et al. 2000, Labrie, Sugimoto, et al. 1992,Labrie, Simard et al, 1992; Luu-The, et a 1995; and Labrie, Durocher etal. 1995).

The data showing the presence of relatively high levels of androgenmetabolites in normal women (Labrie, Manger, et al. 1997; Labrie,Bélanger et al., 2006; Labrie, and Bélanger, et al, 2007b) stronglysuggest that the androgens play a major physiological but stillunderestimated role in women. The 44.5% fall which occurs in serum DEAfrom 20 to 30 years of age to the age of 40-50 years in women (Bélangeret al., 2006) could well explain the bone loss which precedes menopause.In fact, age-related bone loss has been reported to begin in the fourthdecade and changes in bone turnover have been found well beforemenopause (Mazess 1982; Riggs, et al, 1981, and Johnston et al. 1985).In agreement with these findings, bone density was lower at all sitesexamined in women classified as perimenopausal compared to premenopausal(Steinberg, et al., 1989). In agreement with these findings, the changesin precursor androgen secretion by the adrenals precede by 10-20 yearsthe decrease in ovarian estrogen secretion which abruptly stops atmenopause (Labrie, Manger, et al. 2006)

It is important to realize that not only serum DHEA and DHEA-S decreaseby 50% between the ages of 21 years and 50 years but that a similardecrease is observed for serum testosterone (Zumoff et al. 1995). Suchdata could well suggest that hormone replacement therapy with androgensor their recursor(s) should start early at menopause in order tocompensate for this early fall in the secretion of androgen precursorsby the adrenals and the parallel decrease in serum testosterone (Labrie,2006).

The active androgens and estrogens synthesized in peripheral targettissues exert their activity in the cells of origin and very littlediffusion of the active sex steroids occurs, thus resulting in very lowlevels in the circulation. In fact, as observed previously (Labrie,Bélanger et al., 1997) and confirmed in the present study, the moststriking effects of DHEA administration are seen on the circulatinglevels of the glucuronide derivatives of the metabolites of DHT, namelyADT-G and 3 α-diol-C while no significant or only minor changes are seenin the serum levels of testosterone, E1 or E2. These active steroids areproduced locally in the peripheral intracrine tissues which possess theappropriate steroidogenic enzymes to synthesize DHT from the adrenalprecursors DHEA and DHEA-S as well as the enzymes that transform DHTinto the inactive metabolites AU and 3α-diol which are further modifiedby glucuronidation (Manger et al 2003).

In a recent study, daily oral administration of 50 mg of DHEA had nosignificant effect on serum testosterone or DHT while DHEA and ADT-Gwere increased to a similar extent (80-90%) (Arlt et al. 2001).Inanother study, predosing serum levels of DHEA-S in postmenopausal womenwere increased from 0.55 μg/ml to about 1.4 μg/ml (Casson et al 1998),after daily oral administration of 25 mg of DHEA for 6 months. SerumDHEA and testosterone levels, however, measured 23 h after lastadministration of DHEA, were not changed significantly. Another studyhas indicated that the 50 mg daily oral dose of DHEA leads to serumandrogen levels in the premenopausal range (Buster et al. 1992).

The present data clearly demonstrate that DHEA and DHEA-S are convertedin specific peripheral intracrine tissues into active androgens and/orestrogens which can exert their biological effects at their site ofsynthesis with no or only small release of active steroids in thecirculation. Accordingly, changes in serum levels of testosterone, E1 orE2 cannot be used as parameters of transformation of DHEA into androgensor estrogens (Labrie, Bélanger et al., 2006). In fact, the activesteroids are metabolized in the same cells where they have beensynthesized and exerted their action into inactive glucuronidated andsulfated metabolites which finally diffuse in the extracellularcompartment and can be measured in the circulation (Labrie, Bélanger etal., 2006; Labrie, Bélanger et al., 1997 and Labrie, Bélanger et al.2007). Measurement of the conjugated metabolites of androgens is theonly approach that permits an accurate estimate of the total androgenpool in women. It is most likely that a similar situation exists forestrogens, although a precise evaluation of the pharmacokinetics ofestrogen metabolism and identification of their metabolites remains tobe established.

Example 3 Clinical Trail ERC-210 Intravaginal DHEA, the PhysiologicalTreatment of Vaginal Atrophy Subjects and Methods

This study is a phase III, prospective, multicenter, randomized,placebo-controlled, and double-blind trial of 50 subjects per arm (for atotal of 200 subjects). Two hundred postmenopausal women were thusrandomized to receive a daily ovule of the following DHEAconcentrations: 0.0%, 0.25% (3.25 mg DHEA), 0.5% (6.5 mg DHEA) or 1.0%(13 mg DHEA) applied intravaginally with an applicator. The study wasdivided into two phases, namely screening followed by a treatment periodof 12-week duration,

The inclusion criteria were the following:

Postmenopausal women who satisfy either a or b or c:

a. No menses for at least one year, or;b. FSH levels ≥0.40 mlU/mL (within 60 days prior to Day 1) in women withno menses ≥6 months but <12 months, or hysterectomized women who werepremenopausal at the time of hysterectomy or;c. Six weeks or more (of screening visit) following bilateraloophorectomy.

Women who have self-identified at least one moderate to severe of thefollowing symptoms:

Vaginal dryness (none, mild, moderate or severe).

Vaginal and/or vulvar irritation/itching (none, mild, moderate orsevere),

Vaginal pain associated with sexual activity (none, mild, moderate orsevere).

Women should identify which symptom is the most bothersome to her atstart of treatment. The change of this symptom will be followed and willserve to evaluate the effect of treatment.

Women between 40 and 75 years of age.

Willing to participate in the study and sign an informed consent.

Women having a low maturation index (no greater part of guidance than 5%of superficial cells on vaginal smear).

Women having a vaginal pH above 5.

Normal mammography within 9 months of study start.

Normal breast examination.

A normal PAP smear (which includes inflammatory changes) within the last12 months (of Day 1). For hysterectomized women, the PAP smear willconsist of at least one slide.

No former or present narcotic addiction or alcoholism.

Body weight within the range of 18.5 to 35 of ideal body weightaccording to body mass index (BMI)(WHO).

No hepatic or renal impairment or condition known to affect drug orsteroid metabolism.

Normal baseline hematology, clinical chemistry, and urinalysis.

Negative serology for HIV1/HIV2 and hepatitis B and C.

The exclusion criteria were:

Undiagnosed abnormal genital bleeding.

Previous diagnosis of cancer, except skin cancer (non melanoma).

Endometrial hyperplasia at biopsy performed at screening or endometrialcancer.

Active or history of thromboembolic disease.

Significant metabolic or endocrine disease.

Clinically significant gastrointestinal, liver or gallbladder disease.

Recurrent migraine headache not controlled by conventional therapy.

Diabetes mellitus not controlled by conventional therapy.

Significant complication on previous hormonal therapy.

Use of estrogen alone injectable drug therapy or progestin implantwithin 3 months prior to study entry (screening visit).

Use of estrogen pellet or progestin injectable drug within six monthsprior to study entry.

Oral estrogen, progestin or DHEA exposure or intrauterine progestintherapy in the eight weeks prior to baseline assessments.

Vaginal hormonal products (rings, creams or gels) or transdermalestrogen alone or estrogen/progestin products in the 4 weeks prior tobaseline assessments.

Patients can washout as follows, but the questionnaire on vaginalatrophy must be answered after the required washout period:

At least an eight-week washout period for prior oral estrogen, DHEAand/or progestin therapy.

At least a four-week washout period for prior transdermal hormonetherapy

At least a four-week washout period for locally delivered hormonereplacement therapy for vaginal dryness.

At least 6 months for prior estrogen pellet therapy or progestininjectable drug therapy.

Eight weeks or longer for prior intrauterine progestin therapy.

Six months or longer for prior progestin implants and estrogen aloneinjectable drug therapy.

Previous treatment with androgens or anabolic steroids within 3 monthsprior to screening visit.

Oral corticosteroid treatment within six weeks of study start.

No chronic use of corticosteroid allowed (intermittent nasal spray ortopical on skin, eyes or ears is permitted).

Cardiac failure or manifest coronary heart disease.

Hypertension equal to or above 160/95 mm Hg or not controlled bystandard therapy.

Confirmed clinically significant depression or confirmed history ofsevere psychiatric disturbance.

The administration of any investigational drug within 30 days ofscreening visit.

Clinically relevant abnormal serum biochemistry or haematology.

Baseline cervical cytology showing low-grade squamous intraepitheliallesion (LGLSIL) or worse.

Smoking more than 10 cigarettes a day.

Drugs that interfere with the metabolism of estrogens (eg, ketoconazole,steroid formation or action inhibitors).

SERMs or drug interacting with steroid receptors.

Known presence of uterine fibroma or palpable at gynecological exam.

Coagulation disorders or on anticoagulant drug therapy.

Laboratory Tests

The usual laboratory tests, namely hematology (including complete bloodcount and coagulation), blood chemistry and urinalysis were performed atall visits. Serum FSH had to be measured only in women who had no mensesfor ≥6 months but <12 months or who were premenopausal at the time ofhysterectomy. Serum steroid levels of DHEA, DHEA-S, androst-5-ene-3β,17β-diol (5-diol), dihydrotestosterone (DHT), testosterone (testo),androstenedione (4-dione), estrone (E₁), estradiol (E₂), E₁-S,androsterone glucuronide (ADT-G), androstane-3α, 17β-diol-3G(3α-diol-3G) and 3α-diol-17G were measured at the Laboratory ofMolecular Endocrinology, CHUL Research Center by mass spectrometry asdescribed (Labrie, Bélanger et al. 2006; Labrie, Bélanger et al. 2007;Labrie, Cusan et al. 2008),

Vaginal ph and Cytology

For the maturation index and Papanicolaou (PAP) smear, all samples wereexamined by the same cytopathologist (Dr. Robert Dubé, Department ofcytology-pathology, Enfant-Jésus Hospital, Quebec City, Canada) blindedto the treatment regimens. A 100-cell count was performed to classifycells as superficial (S), intermediate (l) and parabasal (P) squamouscell types (Meisels 1967; Wied 1993).

Vaginal smears were obtained by scraping the middle third of the sidewall of the vagina with the rounded end of an Ayre spatula. The materialwas then applied to a glass slide and immediately fixed with Spray-Cyte.These samples were sent to the central laboratory for determination ofthe maturation index.

Vaginal pH was measured by applying a pH indicator strip directly to thelateral wall of the vagina with a forceps. For the Papanicolaou smear—ifnot done in the last 12 months, specimens were obtained from theendocervix, exocervix and vaginal vault and immediately fixed withcytospray. The specimens were collected with an Ayre spatula.

Endometrial Biospy

Endometrial biopsy was performed at screening and at month 3 at end ofstudy. All biopsies were examined by the same pathologist at the centrallaboratory (Dr. Robert Dubé, Department of cytology-pathology,Enfant-Jésus Hospital, Quebec City, Canada).

Vaginal Examination

At the same time intervals of 2, 4, 8 and 12 weeks, the gynecologist orphysician in charge of the study at each site performed a vaginal examto evaluate the degree of severity (none, mild, moderate or severe,analyzed using values of 0, 1, 2 and 3, respectively) for the main signsof vaginal atrophy, namely vaginal secretions, vaginal color, vaginalepithelial integrity and vaginal epithelial surface thickness. As can beseen in FIGS. 26 to 29, a time-dependent dose-related and statisticallysignificant improvement of all four signs of vaginal atrophy was seen.In fact. the beneficial effects observed by the gynecologist and orphysician are almost superimposable to those self-reported by women ontheir most bothersome symptoms.

Vaginal examination was performed at screening and then at day 1 and at2, 4, 8 and 12 weeks. Vaginal secretions, vaginal color, vaginalepithelial integrity and vaginal epithelial surface thickness wereevaluated according to the following degrees of severity: none, mild,moderate or severe. The definitions of severity were as follows:

a) Vaginal Secretions

No atrophy: Normal clear secretions noted on vaginal walls.

Mild: Superficial coating of secretions, difficulty with speculuminsertion.

Moderate: Scant not covering the entire vaginal vault, may needlubrication with speculum insertion to prevent pain.

Severe: None, inflamed, ulceration noted, need lubrication with speculuminsertion to prevent pain.

b) Vaginal Epithelial Integrity

No atrophy: Normal.

Mild: Vaginal surface bleeds with scraping.

Moderate: Vaginal surface bleeds with light contact.

Severe: Vaginal surface has petechiae before contact and bleeds withlight contact.

c) Vaginal epithelial surface thickness

No atrophy: Rugation and elasticity of vault.

Mild: Poor rogation with some elasticity noted of vaginal vault.

Moderate: Smooth, some elasticity of vaginal vault.

Severe: Smooth, no elasticity, constricts of the upper ⅓ of vagina orloss of vaginal tone (cystocele and rectocele),

d) Vaginal Color

No atrophy: Pink.

Mild: Lighter in color.

Moderate: Pale in color.

Severe: Transparent, either no color or inflamed.

Statistics

Summary tabulations will be prepared that will display the number ofobservations, mean or geometric mean as appropriate, standard deviation,standard error of the mean, 95% two-sided confidence interval (CI),median, minimum, and maximum for continuous variables, and the numberand percent per category for categorical data. Statistical analyses willbe performed at the two-sided significance level of 0.05 unlessotherwise stated. The categories for summarization will in generalconsist of the dose levels of the DHEA treatments, 0% (placebo), 0.25%,0.5% and 1.0% DHEA.

The primary endpoints for analysis will consist of the following:

Statistically significant improvement in the moderate to severe symptomidentified by the subject as most bothersome to her. The symptomseverity is based on symptoms of increasing severity: none, mild,moderate or severe. These ratings will be analyzed using the values 0,1,2 and 3, respectively; all subjects must have at least one baselinesymptom that is graded as 2 or 3. The symptoms of interest are vaginaldryness, vaginal and/or vulvar irritation/itching, and vaginal painassociated with sexual activity.

Statistically significant decrease in parabasal cells and astatistically significant increase in superficial cells. The data ismeasured in percentage. The maturation value will also be calculated.

Statistically significant lowering of vaginal pH.

Analysis Populations

The Intent-to-Treat (ITT) Population will consist of the treatedsubjects with a baseline and at least one post-baseline efficacyassessment. Subjects who may have received the wrong treatment will beanalyzed as randomized. This analysis population is to be considered theprimary analysis population. Subjects in this population who are missingobservations post-baseline will have the last value carried forward forefficacy analyses.

The Per Protocol (PP) Population consists of the subset of the treatedpopulation that completes the study through the time point of 12 weekswith no major protocol violations considered to compromise efficacydata. Major protocol violations will be determined before the studyblind is broken, based on review of data listings and monitoring reportsof protocol deviations. Subjects in the PP population must have receivedat least 90% of the required number of applications of study treatmentin the protocol-specified duration for that subject, based on thesubject diary data. Subjects in the PP population must be compliant withthe visit window schedule: ±3 days for Day 14, and ±7 days for Weeks 4,8 and 12. Subjects who received the wrong treatment, but for whom thetreatment received can be unequivocally confirmed, will be analyzed inthe PP Population as treated, provided they have no other violationsthat compromise their data. The PP population will be a supportivepopulation for efficacy data analysis.

The Safety Population will be defined as all subjects who receive anadministration of either test article (DHEA at any dose or Placebo), andwho have any safety information available. All safety data analyses willbe based on this population. Analysis will be based on the treatmentactually received.

Efficacy Evaluation

Efficacy analyses will be performed primarily on the ITT Population, andthe Per-Protocol population will provide supportive efficacy analyses.The primary study objective is to evaluate the dose-response of vaginalmucosal parameters to the local action of DHEA in postmenopausal womensuffering from vaginal atrophy, specifically by determination of theminimal dose of DHEA that produces maximal effect on the vaginal mucosa.The co-primary efficacy endpoints to address this objective are decreasein parabasal cells, decrease in vaginal pH, increase in superficialcells (collectively, these endpoints will be denoted as physiologicalparameters) and subject self-reported most bothersome symptom includingvaginal dryness, vaginal and/or vulvar itching/irritation, and vaginalpain associated with sexual activity (collectively, these endpoints willbe denoted as symptom score parameters). In addition to these primaryendpoints, the maturation value will also be calculated. Theself-reported symptom scores take the following values: none, mild,moderate or severe to be analyzed using values of 0, 1, 2 or 3,respectively. All endpoints must demonstrate statistically significanteffects relative to placebo, therefore no statistical adjustment isrequired for multiple endpoints.

The primary timepoint for analysis will be the 12-week assessment, withadditional presentations of the data for 2, 4 and 8 weeks. The changefrom baseline to post-baseline assessment will be used for analysis aswell as the difference with placebo.

Results

Since parabasal cells are usually the predominant category in thevaginal smear of postmenopausal women suffering from at least onemoderate to severe symptom of vaginal atrophy, it can be seen in FIG. 22and Table 6 that already at 2 weeks of treatment, the lowest dose ofDHEA (0.25%) decreased the % of parabasal cells by 29.5±0.51% from 56.0to 26.5% while decreases of 37.8±0.46% and 36.6% were observed,respectively, with the 0.5% and 1.0% DHEA doses at the same timeinterval. At the standard duration of 12 weeks of treatment, decreasesof 39.5±0.57% (p<0.000001), 45.6±0.55% (p<0.000001) and 45.2±10.53%(p<0.000001) were observed with the 0.25%, 0,5% and 1.0% DHEA doses,respectively, while no significant effect was observed in the placebogroup at any time interval.

While no significant effect was seen at 12 weeks in the placebo group onthe % change in superficial cells (able 6), increases of 3.% 0.10%(p=(0.0002), 6.71±0.14% (p=0.00001) and 592±0.12% (p=0.00001) weremeasured in the 0.25%, 0.5% and 1.0% DHEA groups, respectively. It canalso be seen that at the 0.5% DHEA dose, 48.0% of the maximal effect wasreached at 2 weeks while at 4 and 8 weeks, 84.8% and 99.0% of themaximal effect were achieved. At the 1.0% DHEA dose, the maximal effectwas already reached at 2 weeks. FIG. 23 illustrates the absolute valuesof the % of superficial cells at the different DHEA doses and timeintervals.

Vaginal pH was decreased at 12 weeks by 0.47±0.11 from 6.52±0.13 unitsin the placebo group (Table 6, FIG. 24) while decreases of 1.12±011(p=0.0005) from 6.49±0.12 units, 1.35±0.3 from 6.56±0.13 pH units,1.35±0.13 from 6.56±0.13 pH units and of 1.39±0.14 from 6.34±0.3 pHunits were observed in the 0.25%, 0.50% and 1.0% DHEA-treated groups,respectively (Table 7). t can be seen in the same table that at the 0.5%DHEA dose, 70.6% and 94.1% of the maximal effect on pH (reduction of1.36 pH units) was achieved at 2 and 4 weeks of treatment, respectively.The low 0.25% DHEA dose, on the other hand, reached only 83.0% of themaximal effect of 0.5% DHEA at 12 weeks. No significant difference inthe change of pH was observed between the 0.50% and 1.0% DHEA doses at4, 8 and 12 weeks (Table 7). FIG. 24 illustrates the absolute pH valuesat the different DHEA doses and time intervals.

All women needed to have at entry one or more of the following symptomsof vaginal atrophy evaluated by herself as moderate to severe: dryness,vaginal or vulval irritation/itching or vaginal pain at sexual activity.The self-identified symptoms reported as none, mild, moderate or severewere analysed using values of 0, 1, 2 and 3, respectively. Asillustrated in Table 8, at the 12-week interval, the severity of themost bothersome symptom was reduced by 0.67±0.15 in the placebo group,1.27±0.16 in the 0.25% DHEA group (p=0.004 vs placebo), 1.56±0.15 in thegroup receiving 0.5% DHEA (p<0.0001 vs placebo) and 1.37±0.14 in thegroup receiving the higher 1.0 DHEA dose (p=0.0008 vs placebo). FIG. 25illustrates the degree of improvement of the most bothersome symptom atthe different DIEA doses and time intervals. Vaginal dryness, painassociated with sexual activity and vaginal and/or vulvarirritation/itching were identified at baseline as the most bothersomesymptom. In the placebo group, vaginal dryness accounted for % of theimprovements noted by the participants.

As illustrated in FIG. 25, the improvement of the most bothersomesymptom was already highly significantly different (p=0.004) at the0.25% DHEA dose. The percentage of women with no change or a worseningof a score of 1 at 12 weeks went from 53.5% in the placebo group to27.5%, 17.8% and 19.6% in the 0.25%, 0.5% and 1.0% groups, respectively(Table 9). The improvements by 2 or 3 categories of severity wereobserved in 21.8% of women treated with placebo while 50.0%, 53.3% and47.9% of women who received the 0.25%, 0.3% and 1.0% DIEA formulationsreported such an important improvement. Only 4.6% of women indicated adecrease from severe to none in the placebo group compared with 7.5%,20% and 10.9% in the same DHEA-treated groups.

At the same time intervals of 2, 4, 8 and 12 weeks, the gynecologist orphysician in charge of the clinical trial at each study site performed avaginal exam to evaluate the degree of severity (none, mild, moderate orsevere analyzed using values of 0, 1, 2, and 3, respectively) for themain signs of vaginal atrophy, namely vaginal secretions, vaginal color,vaginal epithelial integrity and vaginal epithelial surface thickness.As can be seen in FIGS. 26 to 29, a time-dependent and dose-related aswell as highly statistically significant improvement of all four signsof vaginal atrophy was seen. In fact, the beneficial effects observed bythe gynecologist or physician are almost superimposable to thoseself-reported by women on their most bothersome symptoms as well as tothe effects on vaginal parabasal and superficial cells and pH which areobjective parameters of DHEA action.

FIGS. 30 and 31 illustrate the average 24 h (calculated fromAUC_(0-24 h) values measured on days 1 and 7 of treatment) serum steroidlevels of DHEA and eleven of its metabolites taken from a recent study(Labrie, Cusan et al. 2008). It can be seen that only serum DHEA and5-diol (and 4-dione at day 1) are increased significantly but wellwithin the limits of values found in postmenopausal women (Labrie,Banger et al. 2006). Serum estrogens (E₁, E₂ and E₁S) as well as serumandrogens (testo and DHT) are not significantly affected.

TABLE 6 Change from day 1 in % parabasal and superficial cells duringlocal treatment with increasing doses of DHEA* 2 weeks 4 weeks 8 weeks12 weeks Parabasal cells  0.0% DHEA  +3.6 ± 0.32 +0.02 ± 0.32 +1.17 ±0.37 +1.04 ± 0.35 0.25% DHEA −29.5 ± 0.51 −38.4 ± 0.51 −40.3 ± 0.55−39.5 ± 0.57 0.50% DHEA −37.6 ± 0.46 −43.4 ± 0.50 −47.6 ± 0.49 −45.6 ±0.55  1.0% DHEA −36.6 ± 0.50 −42.5 ± 0.51 −43.7 ± 0.50 −45.2 ± 0.53Superficial cells  0.0% DHEA  0.10 ± 0.03  0.37 ± 0.03  0.40 ± 0.03 0.53 ± 0.05 0.25% DHEA  2.32 ± 0.07  3.38 ± 0.08  3.42 ± 0.09  3.96 ±0.10 0.50% DHEA  3.22 ± 0.05  5.69 ± 0.09  6.64 ± 0.11  6.71 ± 0.14 1.0% DHEA  6.26 ± 0.16  6.64 ± 0.14  6.88 ± 0.16  5.92 ± 0.12 *mean ±SEM

TABLE 7 Change from day 1 in vaginal pH during local treatment withincreasing doses of DHEA* DHEA dose 2 weeks 4 weeks 8 weeks 12 weeks 0.0% DHEA  −3.6 ± 0.08 −0.37 ± 0.09 −0.51 ± 0.10 −0.47 ± 0.11 0.25%DHEA −0.76 ± 0.12 −0.93 ± 0.12 −1.09 ± 0.10 −1.12 ± 0.11 0.50% DHEA−0.96 ± 0.14 −1.28 ± 0.12 −1.36 ± 0.12 −1.35 ± 0.13  1.0% DHEA −1.13 ±0.12 −1.30 ± 0.12 −1.41 ± 0.12 −1.39 ± 0.14 *mean ± SEM

TABLE 8 Change from day 1 in the most bothersome symptoms of vaginalatrophy during local treatment with increasing doses of DHEA* DHEA dose2 weeks 4 weeks 8 weeks 12 weeks  0.0% DHEA −0.49 ± 0.16 −0.79 ± 0.16−0.61 ± 0.16 −0.67 ± 0.15 0.25% DHEA −0.70 ± 0.17 −1.11 ± 0.16 −1.19 ±0.16 −1.27 ± 0.16 0.50% DHEA −0.98 ± 0.15 −1.36 ± 0.15 −1.37 ± 0.17−1.56 ± 0.15  1.0% DHEA −1.00 ± 0.15 −1.29 ± 0.14 −1.38 ± 0.17 −1.37 ±0.14 *mean ± SEM

TABLE 9 Change from day 1 in the most bothersome symptoms at 12 weeks oftreatment with 0% (placebo), 0.25%, 0.50% and 1.0% DHEA. Change from onecategory (Severe → moderate → mild → none) was taken as −1 while achange of 2 categories was −2, etc . . . Category change −3 −2 −1 0 +1Doses % woman  0.0% DHEA 4.65 16.3 25.6 48.8 4.65 0.25% DHEA 7.50 42.522.5 25.0 2.50 0.50% DHEA 20.0 33.3 28.9 17.8 0.0  1.0% DHEA 10.9 37.032.6 17.4 2.17

Set forth below, by way of example and not of limitation, are severalpharmaceutical compositions utilizing preferred active sex steroidprecursor DHEA. The concentration of active ingredient may be variedover a wide range as discussed herein. The amounts and types of otheringredients that may be included are well known in the art.

Example A

Vaginal or oral Tablet Weight % Ingredient (by weight of totalcomposition) DHEA 5.0 Gelatin 6.5 Lactose 70.5 Starch 18.0

Example B

1.3 ml Vaginal suppository Weight % Ingredient (by weight of totalcomposition) DHEA 0.50 Whitepsol H-15 base 99.50DHEA suppositories were prepared using Whitepsol H-15 base (availablefrom Medisca, Montreal, Canada). Any other lipophilic base such as butnon limited to butter, cocoa butter, Cotomar, Dehydag Base, Fattibase,Hexaride Base 95, Hydrokote, Suppocire, Wecobee, theobroma oil,Japocire, Ovucire, Massa Estarinum or other combinations of theforegoing could used.

Example C

Vaginal or topical cream Weight % Ingredient (by weight of totalcomposition) DHEA 1.0 Emulsifying Wax, NF 18.0 Light mineral oil, NF12.0 Benzyl alcohol 1.0 Ethanol 95% USP 34.0 Purifed waer, USP 34.0

Vaginal or Oral Gelatin Capsule

Other sex steroid precursors may be substituted for DHEA in the aboveformulations. More than one precursor may be included in which case thecombined weight percentage is preferably that of the weight percentagefor the single precursor given in the examples above.

The invention has been described in terms of preferred embodiments andexamples, but is not limited thereby. Those of skill in the art willreadily recognize the broader applicability and scope of the inventionwhich is limited only by the patent claims herein.

1. A method of treating and/or reducing the likelihood of acquiringsymptoms or diseases due to the menopause, in postmenopausal women, saidmethod comprising administering a sex steroid precursor selected fromthe group consisting of dehydroepiandrosterone,dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol, and4-androsten-3,17-dione to a patient in need of said treatment whereinthe said sex steroid precursor is administered at a therapeutic amountwhich increases the level of circulating androgen metabolites withoutincreasing the circulating level of estradiol above the values found innormal postmenopausal women in order to avoid the risk of breast anduterine cancer.
 2. The method of claim 1 wherein the sex steroidprecursor is administered on the skin.
 3. The method of claim 1 whereinthe sex steroid precursor is administered intravaginally.
 4. The methodof claim 1 wherein the sex steroid precursor is administered orally. 5.The method of claim 2 wherein the therapeutic amount of sex steroidprecursor is 200 mg per day or less.
 6. The method of claim 2 whereinthe therapeutic amount of sex steroid precursor is 100 mg per day orless.
 7. The method of claim 2 wherein the therapeutic amount of sexsteroid precursor is 30 mg per day or less.
 8. The method of claim 2wherein the therapeutic amount of sex steroid precursor is 15 mg per dayor less.
 9. The method of claim 3 wherein the therapeutic amount of sexsteroid precursor is 50 mg per day or less.
 10. The method of claim 3wherein the therapeutic amount of sex steroid precursor is 25 mg per dayor less.
 11. The method of claim 3 wherein the therapeutic amount of sexsteroid precursor is 13 mg per day or less.
 12. The method of claim 3wherein the therapeutic amount of sex steroid precursor is 6.5 mg perday or less.
 13. The method of claim 4 wherein the therapeutic amount ofsex steroid precursor is 200 mg per day or less.
 14. The method of claim4 wherein the therapeutic amount of sex steroid precursor is 100 mg perday or less.
 15. The method of claim 4 wherein the therapeutic amount ofsex steroid precursor is 50 mg per day or less.
 16. The method of claim1 wherein the average 24 h-level of circulating estradiol is below 10μg/mL.
 17. The method of claim 2 wherein the therapeutic amount of sexsteroid precursor is administered by means of a topical formulationselected from the group consisting of cream, lotion, gel, ointment,sustained release patch, and the like, and wherein said formulationcontains 10% of sex steroid precursor or less.
 18. The method of claim 2wherein the therapeutic amount of sex steroid precursor is administeredby means of a topical formulation selected from the group consisting ofcream, lotion, gel, ointment, sustained release patch, and the like, andwherein said formulation contains 2.0% of sex steroid precursor or less.19. The method of claim 3 wherein the therapeutic amount of sex steroidprecursor is administered by means of a intravaginal formulationselected from the group consisting of cream, lotion, gel, ointment,ovule, suppository, ring, and the like, and wherein said formulationcontains 10% of sex steroid precursor or less.
 20. The method of claim 3wherein the therapeutic amount of sex steroid precursor is administeredby means of a intravaginal formulation selected from the groupconsisting of cream, lotion, gel, ointment, ovule, suppository, ring,and the like, and wherein said formulation contains 2.0% of sex steroidprecursor or less.
 21. The method of claim 4 wherein the therapeuticamount of sex steroid precursor is administered by means of an oralformulation selected from the group consisting of capsules, plugcapsules, pills, tablets, syrups.
 22. The method of claim 1, furthercomprising administering as part of a combination therapy, atherapeutically effective amount of a Selective Estrogen ReceptorModulator.
 23. A method of treating and/or reducing the likelihood ofacquiring symptoms or diseases due to the menopause, in postmenopausalwomen, said method comprising administering a sex steroid precursorselected from the group consisting of dehydroepiandrosterone,dehydroepiandrosterone-sulfate, androst-5-ene-3β,17β-diol, and4-androsten-3,17-dione to a patient in need of said treatment whereinthe said sex steroid precursor is administered at a therapeutic amountwhich increases the level of circulating androgen metabolites andfurther comprising administering as part of a combination therapy, atherapeutically effective amount of a Selective Estrogen ReceptorModulator in order to decrease the risk of breast and uterine cancernormally present in postmenauposal women and to prevent bone loss, fataccumulation and diabetes type
 2. 24. The method of claim 23 wherein theSelective Estrogen Receptor Modulator has the following chemicalstructure.


25. The method of claim 1 wherein the symptoms or diseases due to themenopause are selected from the group of diseases consisting ofosteoporosis, vaginal atrophy, vulvo-vaginal dryness, hypogonadism,diminished libido, skin atrophy, connective tissue disease, urinaryincontinence, loss of muscle mass, insulin resistance, fatigue, loss ofenergy, aging, and physical symptoms and signs of menopause. 26-38.(canceled)
 39. The method of claim 23 wherein the symptoms or diseasesdue to the menopause are selected from the group of diseases consistingof osteoporosis, vaginal atrophy, vulvo-vaginal dryness, hypogonadism,diminished libido, skin atrophy, connective tissue disease, urinaryincontinence, loss of muscle mass, insulin resistance, fatigue, loss ofenergy, aging, and physical symptoms and signs of menopause.